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The back to school season has returned, which also means your busy schedule is coming back. How can our local storage in Ellijay GA help you prepare for this upcoming school year? By providing you extra space, all your seasonal gear will be safely secured until next season. Also, we have tips for your house that will help get you and your children ready for the school year. Take a look at some of the ways our local self storage units help with the back to school season! HELPFUL ORGANIZATION AROUND YOUR HOME Arranging the family's schedule and planning meals is all part of the back to school routine. Use these tips to help get organized quick! Create a homework center. It can be the kitchen table or in an office space, as long as your child has a quiet place to be able to concentrate on their school work. Take inventory of your kid's clothes. Go through their closets and drawers and get rid of any articles of clothing that no longer fit them. You can then make a list of what they need and go shopping so you are not rushing around last minute trying to find clothes. Download a family calendar app (Cozi Family Organizer) so you can see everyone's schedule, which is color-coded for you. Also, you are able to share reminders and there is even an editable shopping list feature. Plan your dinners ahead of time. You can save time and money by prepping and planning meals in advance. This eliminates ordering take out if you are prepared. Have a school supply stash. If your child has a project and you run out of a material they need and it is the evening when stores are closed, then you will have all the items you need. Also buying in bulk can save you cash! Discover many more helpful tips for arranging your home here! HOW TO STORE OUTDOOR EQUIPMENT When its time to put the peddle bikes away there are just a few precautions you ...
September 11th, 2019
Although the population of Ellijay GA may be under 2,000 residents, this city is one gorgeous place to visit. With multiple major parks and mountains to hike, beautiful views are wide spread throughout this community. We at Ellijay Storage are extremely ecstatic to continue to be able to call our Ellijay GA community home. During the month of August we are focusing on what truly makes our neighborhood so special. With varieties of sites to observe, savory food to try, and considerate businesses, these are just some factors making up what we love about our city. DOWNTOWN ELLIJAY GA Experience fantastic restaurants, taverns, live music and shopping in our old fashioned mountain town. Being in a old-school city, many antique shops line the streets downtown. This allows you to stumble upon unique merchandise you can not just find anywhere. Lets check out where our store manager at Ellijay Storage recommends exploring. Back Porch Bistro Cornerstone Cafe Moobears Ice Cream & Hotdogs Mountain Treasures Black Bear Antiques Belle's ACTIVITIES AND APPLES Summer time is Ellijay GA is meant for the wonderful people of this city. Everyday from the beginning of the summer, until the first week of October there is a farmers market open. With enormous amounts of fresh vegetables and homemade crafts, the market is a summer staple in this community. Not only is there the farmers market, but there is an abundance of other activities for the kids throughout the summer to partake in as well. For instance there is: Water Sports Horseback Riding Hiking Ellijay GA is located within Gilmer County GA. For those familiar with the area then know that this county is the "Apple Capital of Georgia". Each year in October the city of Ellijay hosts the annual Georgia Apple Festival which takes place in October. Our manager at Ellijay Storage has said, " They make so many different things from the apples. I never know what to try next."Our Ellijay GA community is one of the states top visited cities year in and year out. Ellijay Storage is ...
August 7th, 2019
Have you ever been curious how some extra space could go a long way for you? Ellijay Storage has 10 reasons how our Ellijay GA affordable storage opportunities can aid you no matter your situation. Having the knowledge of when storage is most beneficial to you can really go a long way. Especially for the time you need extra storage space in a potentially hectic time. Check out our 10 reasons below to see when and how storing at Ellijay Storage can guide you to finding the space you need. YOU MAY NEED STORAGE IF… Selling your home and moving (out of state or in-state). Renovating your home (i.e: bedroom, family room, den, garage, etc.) Downsizing yours or a family members home. Storing hobby or sport equipment. Storing seasonal items and decorations. Children returning from college for the summer. Recent change in your relationship status. Starting a online business. Storing extra work equipment and inventory. Traveling for work. VALUABLE FEATURES Options in relation to self storage are basically endless. That is why we at Ellijay Storage administer storage features that our customers want and also benefit from. Having convenient online bill paying allows you to not have to worry about making it in the office during the allotted times. Also with Ellijay storage we have four locations (annexes) to better help serve our Ellijay GA customers.Ellijay Storage is set on striving to continue to be more that just your storage provider in Ellijay GA. Our facility is safe and secure and able to be used for storing your belongings, whether you are moving into a new home all the way to just needing extra storage space for items that do not always have the room needed at your residency. Come check out our broad selection of neighborhood companions, moving and storage supplies, storage tips and Ellijay GA affordable storage all at Ellijay Storage.If you potentially are moving in the upcoming future or want some extra storage space for your personal belongings, get in contact with our storage professionals at Ellijay Storage. Our storage ...
July 15th, 2019
Preparing for your moving day? You already bought all your moving supplies and packing is underway, but who is going to be the ones helping you on moving day? Yes, friends and family are obviously great help, but now is the time to see if professionals are the right choice. Ellijay Storage has the must ask questions for any Ellijay GA moving company you are planning to hire. Deciding on getting professional movers can reduce some stress of moving. Similar to how storage does for our customers.This month Ellijay Storage has all the moving tips and question you will want to ask your desired moving companies. Check out a Ellijay GA moving company on our neighborhood friends board. Ellijay Storage has a secure storage unit close to home so you can continue to be on track when space is tight during moving. With steady neighborhood friends and self storage experts at Ellijay Storage you will be on the right path for moving day. QUESTIONS TO ASK MOVING COMPANIES Who can I contact on moving day if I have questions? What is your cancellation policy? Are there additional charges for evenings/weekends? How you protect my belongings How do train your staff? Find more information for selecting a moving company and potential questions you may want to ask here.The journey to finding the right moving company for your upcoming move does not have to be strenuous anymore. Ellijay Storage neighborhood friends board is full of Ellijay GA businesses that will be great service to you when moving. Marshall Garrett Moving Services who are loyal movers are just one connection made within the area.Marshall Garrett Moving Services are specialist ready to assist during your big day. Neighboring Blue Ridge GA, Blairsville GA, Dawsonville GA, Canton GA, or Chatsworth GA? Then Marshall Garrett Moving Services is the nearby choice for people moving to the Ellijay GA area. Explore additional Ellijay GA moving services and local places of interest on our website.Can storage near Ellijay GA benefit you? Ellijay Storage knows the ...
June 21st, 2019
Make moving easy with Ellijay Storage! Summer moves, especially long distance moves, can be overwhelming. With the right tips, tools, and space, you can move like a pro. This month Ellijay Storage is preparing for big moves out of state with a cross country checklist.How can storing in Ellijay GA help while moving? Storage in Ellijay GA makes it easy to find a place to store a few boxes while you stage and get organized for an upcoming move. Check out how we are getting you ready for long distance moves with this checklist. A MONTH BEFORE Get quotes from moving companies and storage facilities. We keep our storage prices competitive with the rest of the market. Research the neighborhood or state you are moving to. Schools for the kids, and a few restaurants or businesses will help you feel right at home. If moving near our Ellijay GA neighborhood, check out our neighborhood friends board for businesses we trust! MOVING AND STORAGE TIPS Find packing supplies in our office! We have more than just storage units for sale, we have boxes, tape, storage locks, and more! Notify doctors, dentists, employers, schools, etc of your move. Here is a full list to help you remember everyone. Forward your mail online or fill out the packet from the post office. If you are moving to the area for work or taking on a new beginning with your family, stop by Ellijay Storage for space saving tips. You can rent your next storage unit online, pay your bill, and research which storage unit is best for you, all online! ...
May 24th, 2019
Moving out of the dorm room, mean packing all your gear up, but where do you store it? Summer break is coming fast and Ellijay Storage can help you prepare with our Ellijay GA summer storage. Our storage units are both large and small, so you can choose to store with a friend or store by yourself. Choosing a short term storage unit in Ellijay GA may just be the solution to your summer break.Ellijay Storage may be close to campus or close to home, either way moving your school supplies somewhere safe will make the fall semester that much more relaxing. SUMMER STORAGE TIPS FOR STUDENTS Plan to move the mini fridge a few days before you need to be out of the dorm. Take any leftover food out and leave it unplugged with a towel underneath the open door. Friends or roommates are great to store with! Just remember to use labels and communicate with everyone when it is time to move out. Use our easy pay option to set up your online account and pay your bills with this optional automatic paying option. You can even pay for the whole summer storage up front. Selling your textbooks back to the campus store or to another student may be the best option. You can save space at the storage unit and replenish what you paid at the start of the year. Let us know when you need storage. We can reserve your storage unit when you need it and check this off your list. For more easy to follow packing tips, check out the latest from BuzzFeed. These tips will be helpful if you are packing up your dorm room or moving to a new house.Ellijay Storage is ready to help you store your belongings this summer. Whether it is your dorm room items or seasonal items you need out of the house, we have a secure storage unit to assist. Choose your storage option online and use our online tools to help organize your ...
April 17th, 2019
If you have gear you want to sell online, how can storage help? Ellijay Storage has a storage unit to help you store used items for sale. With our extra space you can enjoy an organized home while making a little extra income.Ellijay Storage offers drive up self storage and temperature controlled storage so you choose what storage unit is right for you. Need help deciding? We have storage experts at our office on 1325 Orchard Road and online resources to answer some of your frequently asked questions about storage. TIPS FOR SELLING ONLINE Selling furniture? Take a picture of each side and add a detailed description for each one. If your small business is starting an online store, be professional with all your customers. Reviews are asked for both buyers and sellers, so your reputation online matters. Name brand items sell faster with an accompanied picture of tags. Look for tags on the inside of a purse, or the tongue of shoes. A few scratches or parts needing replaced, does not stop customers from buying. Just be honest about the wear and tear. SPRING CLEANING: WHAT TO SELL ONLINE? Gaming systems DVDs / CDs Clothing Wedding Decor Choose a website suitable for items for sale. Facebook Marketplace, Etsy, EBay all have a variety of categories but just posting on one site limits your viewers, so post on multiple.In between spring cleaning and your next sale online, use Ellijay Storage to keep all your gear safe. Also, use our storage during your move or home improvement project. Talk with experts or rent online today! ...
March 21st, 2019
Storage for the house too small for your seasonal gear? Ellijay Storage has storage space to make it easy for storing your belongings. From extra furniture to a specific hobby, a storage unit may be just what you need. This month we are focused on small home storage tips! Check out how our Ellijay GA storage may be the right choice for you!Ellijay Storage has 4 locations in Ellijay GA to better serve our customers. We offer RV parking, large drive up storage units, and experts to guide you through your next storage rental. Start with these storage tips for your home, then speak with us about your next steps. HOME STORAGE TIPS FOR A SMALL SPACE Once the family starts to grow many parents use self storage to stay organized. Whether you are getting ready for a new baby or expecting your college student home, self storage can be a solution. Went far away for college? Instead of traveling back home with all your school supplies, use our self storage for summer break. All of our leases are on a monthly basis, so there is no long term commitment. If your kids moved out and you are ready to move into a smaller place, self storage is the home away from home your valuables. Having an online yard sale is very popular now. If you made a business online like Etsy or EBay, make self storage part of your business plan. Use it for products or supplies. Swap your seasonal gear fast! Our easily accessible storage units make getting your items simple. Ellijay Storage can be the space saving solution you need. These home storage tips and our storage experts are the perfect pair to get your home in order. Start renting today by choosing a storage unit online! SEARCHING FOR A NEW HOME? Use sites like Zillow and Trulia to check out new listings before the open house. Your family can look through pictures and see the asking price before your visit.Ellijay Storage wants to make a ...
February 7th, 2019
How can our self storage help you jumpstart your new year? Find extra space for your seasonal items and vehicles, RVs, and boats at Ellijay Storage. Our Ellijay GA storage units are secure and ready to help you get organized for the new year. Along with our storage space and these podcasts you have all the storage tips you need.Another useful tool to use while cleaning your home or business are podcasts. Between scheduling family events and tasks, sitting down to read a self help book about organization can be tough to do, this is where podcasts come in handy. HOME AND ORGANIZATION PODCASTS Change your mindset about organizing with Simplified Organization. This online series will help you maintain the same level of clean space all year long as if it were just cleaned. Move extra boxes, decorations, and memories to storage and create an open organized home!Did you know you can learn how to better your business with a podcasts? Smart Passive Income teaches followers how to manage money, and make extra funds. Business coach, Pat Flynn, answers questions from real businesses and gives solutions.Listen while you run errands or move; podcasts are the new fast solution to self help! Interesting listens such as The Jillian Michaels Show keep you motivate and healthy. Take these tips and be proactive about being healthy.Our Ellijay GA storage space is ideal for storing personal items and business needs. If you are trying to get organized, talk with a storage professional in Ellijay GA. ...
January 1st, 2019
Ellijay GA holiday season is upon us and here at Ellijay Storage we have all the extra storage space you need. From moving supplies for all your fragile items, to local donation drives impacting our community, we are ready for the busiest time of year. Ellijay Storage has drive up self storage units and vehicle parking so you can store all your items with us. Self storage has many benefits and one our customers enjoy the most is peace of mind. We want to help you have the same peace of mind, so give us a call or rent your storage unit online.After all your summer gear is packed away, get ready for the holidays in Ellijay by donating to our initiative. This year we are collecting new unwrapped toys, for Toys for Tots. HISTORY OF TOYS FOR TOTS Toys for Tots started in 1947 with the first toy drive in Los Angeles. Major Bill Hendricks gave the handmade dolls his wife made to children in need and they sparked a community. Bill Hendricks was a Major in the U.S. Marine Corps and thought it would be fitting to have the reserve Corps go out in the community as stand up citizens. The goal was to provide at least one toys for each boy and girl, so they have a gift to open and a role model to look up to.While Toys for Tots is heard of most often during the holidays season, the organization has expanded their efforts to after school programs and supply drives all year long. AFTER THE HOLIDAYS Stop by Ellijay Storage after the holidays are over for organization ideas during the new year. With a fresh start to a new year, clean up your space with our self storage possibilities. Contact the storage experts at Ellijay Storage and make your Ellijay GA holiday organized! ...
November 2nd, 2018


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Wise Staff of life visitors are usually professionals within to save many shelling out less. You get experience to store clever, the right way to grow a budget, and in what way to find the best deals on essentially anything. But yet what about if you wish to make shed and additionally loosen up? Lifespan, in fact, talks about account balance in addition to developing quite a few hobbies is definitely an essential element of dwelling a happy, healthy life. Doubts some of those interests wasn't for that reason expensive. The good news is, there are plenty of methods to stick true to a person's cheap key points nonetheless rejoice inside the process. The following are 35 approaches to take pleasure from yourself devoid of revealing the bank. Much too apparent? It's possible, but it is low-cost, low-priced, affordable, and also if you like doing it, body fat uses up new material. Get great method to obtain products to learn to read along the The web, purchase widely used training books located at yard sales and then parsimoniousness outlet stores, or maybe take advantage of any complimentary library charge card plus earn the means to access several this game titles in order to the next best-sellers.
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That i for no reason honestly experienced restroom window curtains looking for a dress-debacle into my fifth primary Home-Ec training, but this time, using a girl from my very own, We have all pulled out mom's machine, not to mention I'm just little by little understanding the concepts of learning to make by myself garments. My own just feel dissapointed is usually that Simply put i wouldn't practice it sooner. Restroom window curtains is a marvellously inspiring wall socket along with a wonderful way to be able to manner some tailor-made armoire with regard to a small fraction of the things you had spend buying there are various racks. Plus — this also will be transpire — if you've found yourself in fact efficient at employing shapes, you'll be ready employed your self designs. Step above, Jaclyn Penson!

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September's $90-million-dollar man
Remember the 1970s action series featuring Steve Austin (AKA Lee Majors) as the Six-Million-Dollar man, with bionic limbs and superhuman power allowing him to run up to 60 MPH? The Dallas Cowboys made Ezekiel Elliott, the former Ohio State player, the $90 million-dollar man this month.
Elliott's deal influenced by his 40-day holdout from training camp isn't a bad one given that Jerry Jones, owner of the Cowboys, can afford the tab that guarantees the running back a minimum of $50 million over six-years. Forbes magazine consistently ranks the Dallas Cowboys with a net worth over $5 billion, as the most valuable professional franchise in the world. In fact, the NFL is so rich that half of the franchises listed among the top 50 are American football teams. The other half represents a scattering of MLB, NBA, and Premier League soccer teams (such as Manchester United, Real Madrid, and Barcelona). All of the franchises in the top 50 are worth at least $2 billion.
The fascination with football in Tiffin, Ohio rivals any American city from Tuscaloosa, Alabama and Athens, Georgia to Boston or just about anywhere in the continental states — including Dallas, home of that $90-million-dollar man. There is so much inherently good in supporting football, ranging from road trips to Notre Dame and Ohio State to cheering with Calvert's Seneca Sue or ringing the bell after a Columbian score.
There is so much inherently bad, too. In Mobile, Alabama a few weeks ago, a 17-year-old teen shot nine fans inside a stadium while another high school game in Cincinnati was called shortly after a player head-butted an official.
In Tiffin, there's also a lot of football fans following volleyball with keen anticipation for the Columbian-Calvert matchup ending the regular season in mid-October. Soon after football and volleyball ends, many high school seniors will be visiting prospective colleges with a few seeking an athletic scholarship. Money fuels the potential for corruption and big-time college sports have forever been under the radar to curtail cases of fraud or bribery. The most recent FBI crackdown on college basketball and the Varsity Blues Admissions' scam is hitting the sentencing phase. It's ironic that Chuck Person, a former NBA player (1987 Rookie of the Year) and assistant coach at Auburn received no prison time for accepting $91,500 in bribes to steer recruits to particular basketball programs while celebrity actress Felicity Hoffman is to serve 14 days for her role in paying to have her daughter's SAT score embellished.
Both bribery scandals were connected to Division I colleges. Incidentally, a study of NCAA Division I major infraction cases between 1953 and 2014 revealed 83% were related to football and basketball. However, there are 10 times — if not 50 times — as many minor violations throughout all levels of college sports such as a coach pocket-dialing a recruit outside a permissible contact period or a team traveling with an ineligible player due to a delay in filing paperwork. Although the Ohio State football program had its share of trouble following a 2010 incident when Terrelle Pryor and other players sold their memorabilia, the $90-million-dollar man didn't step on campus until years later.
The felonious crimes painting college athletics as a playground for potential corruption are actually a reflection of the macrocosm of society where only a fraction of bad behavior deemed media-worthy are featured widely enough to influence public perception. The notorious point-shaving scandals in collegiate basketball between the 1950s-90s tarnished the NCAA's reputation for providing an uncompromised system of fair play; however, the number of total cases can barely be counted on two hands. Similarly, the small percentage of bribes floating through the admission offices of Ivy league schools last year and the few dozen basketball departments at D-I power institutions involved in the latest corruption scandal have perpetuated a negative image of college sports as a money-hungry machine. Despite the attention on big time scandals in college sports, the ratings for college football are though the roof this September with no slow-down in sight.
There is also plenty, plenty of good in college football and college sports. Hats off to the University of Alabama experimenting with a loyalty app designed to keep students in the stadium seats longer by incentivizing them with upgrades and increased access to SEC playoff games. Maybe the app and some type of incentive would work to keep students in the seats longer at Frost-Kalnow and Hoernemann stadiums, home of the Tiffin Dragons and Heidelberg Student Princes.
Another novel idea is to see if the $90-million-dollar man, void of any true super-human power, is earning his paycheck with the Cowboys over the next few Sundays. So far, Dallas is 3-1 and the $90-million-dollar man ranks fourth in the NFL in average rushing yards. I wonder if Elliott could out-run Caster Semenya, the mid-distance South African runner and Olympic champion who is missing the world championships in Doha, Qatar this week since refusing injection treatments to lower her natural testosterone level. Whether addressing salaries among NFL players or gender equality among world class athletes, the concept of equity is rationalized in a scientific equation instead of a social issue which automatically draws the line between a winner and loser if there is no parity. Guess Elliot wins and Semenya, unfortunately, loses.
Stay tuned next month for more interesting sport stories from around the globe, around the country, around the state, and right here in Tiffin, Ohio.
Bonnie Tiell is a professor of sports management at Tiffin University.


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Glamping is where stunning nature meets modern luxury. Experiential travel is an authentic way to connect with nature. Together, the experience is about stepping off the beaten path, walking away from superficial tourist activities and embracing an immersive cultural environment.
By pushing themselves out of their comfort zones, experiential travelers experience a shift in perspective that improves the way they connect with the world. This transformational style of travel will enrich a person's life and, ultimately, leave an impression that lasts much longer than the journey itself.
We have two Glamping units on site that are available for rent through out the year. Book Now to see availability and reserve your stay! Or check out our pricing on our Rates page.
No Smoking and Pets Not Allowed in Glamping Tents


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SOMERVILLE, NJ – Six weeks of reflection, "Ashes to Go" for commuters at the train station and a sock collection for those in need are all part of the Lenten season at St. John's Episcopal Church, with services and the distribution of ashes on Ash Wednesday, Feb. 26 at the church
Lent is the 40-day season of reflection and penance that begins on Ash Wednesday and leads to Holy Week and Easter on April 12. The distribution of ashes is conducted annually on Ash Wednesday, signifying the beginning of Lent.
Ashes will be distributed at Holy Eucharist three times on Ash Wednesday: 7 a.m., noon, and 7 p.m.
St. John's will offer Ashes To Go at the eastbound Somerville Train Station from 6:30-8 a.m. Through Ashes To Go, those wishing to receive ashes, but who may not have the opportunity to attend a church service that day, can participate in this holy ritual. This marks the second year offering Ashes To Go following an overwhelmingly positive response in 2019.
Lenten Program
Community members and parishioners are invited participate in a free six-session series of Lenten reflection and action on Thursdays from 6:30-8 p.m. at the church.
Each session will begin with a light meal of soup, bread and water, followed by prayer, a scripture reading, reflection, study questions and song.
Sponsored and led by the St. John's Faith in Action Committee, Bibles and reflection aids from the Center for Christian Ethics at Baylor University will be provided for each evening's study.
Topics are:
Feb. 27: Pray without Ceasing: Following St. Paul's instruction;
March 5: Learning to Pray: Learning from the early Church;
March 12: Praying for a Change: Reconciling tradition and contemporary rationality;
March 19: St. John's Faith in Action Committee's Laundry Love ministry will meet at Wayne's Wash World II, 20 Division St., Somerville – all are invited to take part;
March 26: Sweet Hours of Prayer: Establishing a time of quiet and reflection;
April 2: Our Deepest Prayer: Connecting our waiting with God's waiting.
Free-will donations to support St. John's monthly Laundry Love ministry will be accepted.
Sock Drive
From Ash Wednesday, February 26, to Holy Thursday, April 9, the Episcopal Church Women of St John's will be collecting new socks for all ages – men, women, children, toddlers, newborns. Working with Canon Pollock and local/regional social service agencies, the socks will be distributed directly to those in need. Socks can be dropped off at St. John's.
For more information contact St. John's at 908-722-1250 or visit the website at www.stjohnsomerville.org; for information on the Lenten series call 908-507-8020.
St. John's Episcopal Church is located at 158 West High St. Ample free parking is available in the church lot behind the church or on the streets. The church and parish hall are handicapped accessible.


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Translation elongation is the stage of protein synthesis in which the translation factor eEF1A plays a pivotal role that is dependent on GTP exchange. In vertebrates, eEF1A can exist as two separately encoded tissue-specific isoforms, eEF1A1, which is almost ubiquitously expressed, and eEF1A2, which is confined to neurons and muscle. The GTP exchange factor for eEF1A1 is a complex called eEF1B made up of subunits eEF1Bα, eEF1Bδ and eEF1Bγ. Previous studies have cast doubt on the ability of eEF1B to interact with eEF1A2, suggesting that this isoform might use a different GTP exchange factor. We show that eEF1B subunits are all widely expressed to varying degrees in different cell lines and tissues, and at different stages of development. We show that ablation of any of the subunits in human cell lines has a small but significant impact on cell viability and cycling. Finally, we show that both eEF1A1 and eEF1A2 colocalise with all eEF1B subunits, in such close proximity that they are highly likely to be in a complex.
Citation: Cao Y, Portela M, Janikiewicz J, Doig J, Abbott CM (2014) Characterisation of Translation Elongation Factor eEF1B Subunit Expression in Mammalian Cells and Tissues and Co-Localisation with eEF1A2. PLoS ONE 9(12): e114117. https://doi.org/10.1371/journal.pone.0114117
Editor: Jin-Gui Chen, Oak Ridge National Laboratory, United States of America
Received: September 10, 2014; Accepted: November 4, 2014; Published: December 1, 2014
Copyright: © 2014 Cao et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper.
Funding: The work was supported by Wellcome Trust 094180/Z/10/Z, http://www.wellcome.ac.uk/. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Translation elongation is mediated by a range of factors that are highly conserved throughout evolution and that are generally ubiquitously expressed. Translation elongation factor eEF1A delivers the aminoacylated tRNA to the ribosome; this is a GTP dependent process that is stimulated by a macromolecular complex called eEF1B. In lower eukaryotes eEF1B contains a guanine nucleotide exchange subunit eEF1Bα and a structural subunit eEF1Bγ, while higher eukaryotic cells have another guanine nucleotide exchange subunit eEF1Bδ (we are using the nomenclature proposed by Le Sourd et al ([1]).
eEF1Bα is the smallest subunit of the eEF1B complex and has guanine nucleotide exchange (GEF) activity. The C-terminal domain is considered to be necessary and sufficient for its GEF activity [2], and responsible for the interaction between eEF1Bα and eEF1A, while the N-terminal domain is involved in binding to the N-terminal domain of eEF1Bγ [3]. eEF1Bα has been found essential for cell growth in yeast [4], and mutation of this subunit enhances translation fidelity concomitant with a lower translational efficiency [5]. It is assumed that eEF1Bα promotes nucleotide exchange in eEF1A by disrupting interactions between GDP with the P-loop and switch regions of eEF1A [6].
eEF1Bδ is the metazoan-specific subunit of eEF1B; the C-terminus of eEF1Bδ is homologous with eEF1Bα [7] and contains the domain necessary for nucleotide exchange activity. The N-terminal domain of eEF1Bδ has a leucine zipper motif [8], indicating possible binding of other proteins, but this motif is not involved in the polymerization of eEF1Bδ monomers [9], and the N-terminal domain is not sufficient for the dimerization of eEF1Bδ [10]. eEF1Bδ has been found to exist as different isoforms resulting from alternative splicing, producing protein of around 35 kD. Recent studies have identified another eEF1Bδ protein isoform of around 70–80 kD, termed eEF1BδL. The mRNA encoding eEF1BδL contains an extra exon, exon 3, which is skipped in the mRNA transcripts of other isoforms and is tissue specific, expressed only in brain, spinal cord and testis. This exon encodes a 367-amino-acid long N-terminus, which contains a putative nuclear localization signal at amino acids 86–93 [11]. The resulting isoform is expressed in the nucleus where it participates in the heat shock and stress response [11].
eEF1Bγ is the eukaryotic specific subunit of eEF1B. The N-terminal domain of eEF1Bγ contains a region of homology to the theta class of glutathione S-transferases (GSTs) [12]. The role of eEF1Bγ in translation elongation is not well understood. eEF1Bγ is usually found tightly associated with eEF1Bα and can be isolated from eEF1Bα only under strong denaturing conditions. Research using Artemia showed that the nucleotide exchange rate of eEF1Bα is higher in the presence of eEF1Bγ. eEF1Bγ is also likely to be involved in directing other subunits in the eEF1B complex [13] and to play a role in scaffolding for the eEF1B complex [1] as it is highly associated with membrane and cytoskeleton structures.
Although the components of eEF1B have been reasonably well characterised, and eEF1B is considered to form a reversible macro complex with eEF1A (eEF1H) to mediate the guanine nucleotide exchange on eEF1A, how the three subunits of eEF1B combine and interact with eEF1A remains unclear and there is inconsistency between the models proposed. The first structural model proposed was based on in vitro reconstitution experiments using different combinations of the subunits purified from rabbit liver, as well as published information about eEF1H subunits from Artemia by other groups [14]. They suggested a protomer composed of valyl-tRNA and eEF1H, which were associated through eEF1Bδ. Two such protomers could bind to each other via the leucine zipper motif on the N-terminus of two eEF1Bδ subunits. A subsequent study of Artemia suggested a different structural model wherein eEF1Bγ binds to both eEF1Bα and eEF1Bδ, each of which binds to a eEF1A subunit [15], and further models with different features were proposed by other groups [10],[16],[17],[18].
Although the above models are different from each other, some consistent features emerge. Firstly, it is believed that eEF1Bα and eEF1Bγ are tightly associated and can only be separated under denaturing conditions [19]. Secondly, eEF1Bα and eEF1Bδ show no affinity for each other. Finally, the binding sites of eEF1Bα and eEF1Bδ to eEF1Bγ locate on the N-terminus of the three proteins, while the C-terminus of eEF1Bα and eEF1Bδ harbors the binding sites for eEF1A.
A further complication arises from the fact that eEF1A is found as two isoforms in vertebrates, each encoded by a separate gene and each expressed in different cell types. Whilst eEF1A1 is almost ubiquitously expressed, it is downregulated postnatally in neurons and muscle and replaced by eEF1A2. One of the studies that took eEF1A2 into account was a series of yeast two-hybrid (Y2H) analyses, where the cDNAs of both isoforms of eEF1A and all three eEF1B subunits, were cloned into Y2H expression vectors respectively to map the interaction patterns between the proteins. It was found that in contrast with eEF1A1, eEF1A2 has little or no affinity for eEF1Bα and eEF1Bδ [10], the two eEF1B subunits that have GTP exchange activity. This finding was surprising as the two isoforms of eEF1A would be predicted to have similar abilities to bind to eEF1B, particularly eEF1Bα. The amino acid sequences of the two isoforms of human eEF1A are 92% identical. Comparative three-dimensional models of human eEF1A1 and eEF1A2 on the basis of the crystal structure of homologous eEF1A from yeast show that almost all the residues that differ between eEF1A1 and eEF1A2 are found on one side of the molecule, while the binding sites for eEF1Bα are on the other side [20]. Furthermore, whereas eEF1A1 which binds GTP more strongly than GDP, eEF1A2 shows more affinity for GDP than GTP [21], suggesting that eEF1A2 might be more dependent than eEF1A1 on the presence of a GTP exchange factor. One explanation proposed for the different affinities of the two isoforms of eEF1A to eEF1B was the potential existence of a different GEF for eEF1A2 [10]. Multiple chromosomal isoforms of human eEF1Bα have been identified, one of which transcribes a brain- and muscle-specific cDNA [22]. This expression pattern is in accordance with that of eEF1A2 and was suggested to act as the GEF specifically for eEF1A2. However, as this gene is intronless and absent from mice this seems unlikely to be the explanation [23].
In order to resolve this apparent contradiction, we used proximity ligation assays (PLA) to ask whether both eEF1A2 and eEF1A1 can bind to eEF1B subunits. We also analysed expression and the effects of ablating expression of eEF1B subunits in a range of cell lines and tissues.
Expression analysis in cell lines and tissues
Initially we compared expression of each of the three subunits of eEF1B in a panel of cell lines using Western blots. Twelve cell lines were examined, three of which are untransformed (NIH3T3, Rat2 and HEK293). eEF1Bα was detected in all cell lines but was present at lower levels in the NIH3T3, SHSY5Y, Rat2 and NSC34 cells, all of which are untransformed or neuronal in origin. eEF1Bδ at ∼35 kD was present in all cells but at a low level in Rat2 cells. The other splice form of eEF1Bδ that includes exon 3, at 72 kD (labelled eEF1BδL) was only apparent after long exposures in certain cell lines, notably NSC34, a spinal cord by neuroblastoma fusion line with a neuronal phenotype. The expression pattern of eEF1Bγ in all cell lines tested were similar, with no obvious differences apart from strong expression in HEK293 cells (Figure 1A). In general, all subunits are expressed in the majority of cell lines.
Panel A: Immunoblot of eEF1Bα, eEF1Bδ and eEF1Bγ protein expression in cell lines. GAPDH was used as a loading control. Panel B: Immunoblot of eEF1B subunits in 24-day old mice. Each tissue is shown twice, the left hand sample is from a wild type mouse and the right hand sample from a wasted homozygous mouse. SC = spinal cord. The second row shows a longer exposure of the eEF1Bδ blot, revealing the expression of the longer isoform. GAPDH was used as a loading control. Panel C: Immunoblot of eEF1Bδ in an extended panel of mouse tissues showing brain and testis-expression of the longer isoform (top panel shows a longer exposure). GAPDH was used as a loading control. Panel D: Immunoblot of eEF1B subunits expression in brain (Br) and liver (Li) throughout late embryonic and postnatal mouse development. GAPDH was used as a loading control.
We then went on to look at expression in a range of mouse tissues. Panel B of Figure 1 shows expression in paired samples from wild-type and wasted (eEF1A2-null) mice at 24 days of age. The lack of eEF1A2 has no effect on expression, but it can be seen that expression varies between tissues. Every tissue tested expresses all eEF1B subunits, with particularly high expression seen in pancreas. eEF1Bα is expressed at a low level in brain, spinal cord, heart, lung and muscle. These results are generally consistent with those in RNA expression databases such as those in GEO profiles, which also show widespread expression of all subunits at the RNA level.
eEF1Bδ shows different variants, with the 72 kD form expressed only in brain and spinal cord. Other isoforms cluster around 32 to 38 kD and are most strongly expressed in liver, pancreas, spleen and thymus. Panel C shows expression of the eEF1Bδ isoforms in more detail including a longer exposure of the Western, confirming strong expression of the eEF1BδL form at the protein level in brain, spinal cord and testis.
eEF1Bγ is expressed at a similar level in all the tissues tested, except for muscle, which shows weak expression, and pancreas, where it is highly expressed, as for the other subunits (panel B).
Panel D of Figure 1 shows Western blots for each subunit each in brain and liver from mice at different developmental stages. It can be seen that whereas expression of eEF1Bα is barely detectable before birth, eEF1Bδ is expressed at much higher levels at E15.5 and P2 than at later stages, in both liver and brain. eEF1Bγ shows a similar but less dramatic trend. The eEF1BδL isoform, in contrast, is expressed in brain at all ages but showing a slight increase with age.
We then used immunohistochemistry to compare expression of the subunits in a selection of human and mouse tissues. Figure 2 shows the results obtained in pancreas andbrain. Staining in the pancreas shows similarly high levels of expression of each subunit in both human and mouse, with particularly strong expression throughout the islets. Both eEF1Bα and eEF1Bδ are strongly expressed in neurons, although in the case of eEF1Bδ we are unable to tell whether this is staining is specific for the long isoform, as there are no antibodies that distinguish the two. However, there is little sign of nuclear expression, the reported site of eEF1BδL in cells [11].
Immunohistochemistry of eEF1B subunits in human and mouse brain and pancreas. Proteins detected through primary antibody incubation, HRP mouse + rabbit secondary antibody and subsequent incubation with DAB. Positive signal is indicated by the presence of brown DAB reaction product. Panels show eEF1Bα (b and f), eEF1Bδ(c and g) and eEF1Bγ (d and h) Incubation with secondary antibody only was used as negative control (a and e). Bar (top left micrograph) represents 100 and 50 µm respectively.
Ablation of eEF1B subunits in mammalian cells
The protein expression of each eEF1B subunit and GAPDH from cells transiently transfected with three different siRNAs for each of the eEF1B subunits and a scrambled siRNA were compared by Western blot. All three siRNAs targeting eEF1Bα, eEF1Bδ and eEF1Bγ substantially reduced the respective protein at 72 hours in comparison with cells treated with scrambled siRNA (Figure 3a).
Immunoblots of protein extracts from cell lines after RNA interference. Panel A: eEF1Bα, eEF1Bδ and eEF1Bγ protein level efficiently knocked down by three different siRNAs in HeLa cells 72 h after transfection. GAPDH was used as a loading control. Panel B: eEF1Bα, eEF1Bδ and eEF1Bγ protein level efficiently knocked down by three different siRNAs in HCT116 and DLD1 cells 72 h after transfection. GAPDH was used as a loading control.
These experiments were then repeated in two further cell lines, HCT116 and DLD1 cells (Figure 3b). We also examined whether the ablation of any one subunit affected the expression of the other subunits in these cell lines. The results can be seen in Figure 3b. This showed that siRNAs targeting eEF1Bα consistently ablated expression of the target protein but also showed some variable and inconsistent down-regulation of the other two subunits. Targeting of eEF1Bδ was efficient in each cell line, and had no observable effect on either of the other two subunits. In contrast, ablation of eEF1Bγ consistently resulted in downregulation of both eEF1Bα and eEF1Bδ in all cell lines tested, possibly due to a reduction in stability of the complex.
We then examined the effect of downregulation of eEF1B by RNAi on cell viability using Alamar Blue. The mean of at least three independent experiments was plotted relative to the viability of mock transfected cells. Significance was assessed using t-tests to compare results from cells transfected with eEF1B subunits targeting siRNAs compared to non-targeting siRNA. Knockdown of each eEF1B subunit slightly reduced cell viability, by around 14% (eEF1Bα by 14%, eEF1Bδ by 17% and eEF1Bγ by 10%, when compared with cells transfected with a scrambled siRNA (Figure 4a; p<0.05). In HCT116 cells, eEF1Bα, eEF1Bδ and eEF1Bγ siRNA induced knockdown resulted in a reduction of cell metabolism of over 19% (p<0.001), 12% (p<0.05) and 10% (p<0.05) respectively compared with cells transfected with non-targeting siRNAs). In DLD1 cells this effect was even more pronounced, with a reduction in cell viability of at least 20% when either eEF1Bα or eEF1Bδwas downregulated, and 14% for eEF1Bγ compared to cells transfected with a non-targeting siRNA. As the downregulation of some subunits, notably eEF1Bγ, can affect the expression of the other subunits, it is difficult to impart any specificity to these results, but it is clear that loss of eEF1B has an effect on viability/cell metabolism in a variety of human cell lines.
Panel A: A decrease in cellular metabolism is observed when any of the eEF1B subunit protein level is decreased by siRNAs in HeLa, HTC116 and DLD1 cells. Cell metabolism was assessed by the Alamar blue assay. Data were obtained from the mean of three or more independent experiments in HeLa, HCT116 and DLD1 cells, with more than 10 wells each. Error bars indicate +- SEM; n of wells>10; n = 3−4; *, P<0.05; ***, P<0.001 from non-targeting siRNA. Panel B: Knockdown of eEF1B subunits leads to altered cell cycle profile in three cell lines: representative images of the flow cytometry analysis. Error bars indicate +- SEM; n = 3; *, P<0.05; **, P<0.01 of non-targeting siRNA.
Given the links between the cell cycle and eEF1B subunits and the reduction in viability observed when eEF1B subunits are depleted, we went on to examine the cell cycle profile of cells in which eEF1B subunits had been downregulated by RNAi. Cells were collected 72 hours after transfection, stained with propidium iodine and analysed by flow cytometry. Figure 4b shows graphs representing the mean of three independent experiments in which depletion of eEF1B subunits was confirmed by Western blotting.
Depletion of eEF1B subunits by RNAi in HeLa cells increased the proportion of cells in G0/G1 phase by 7%, with a concomitant decrease in cells in S-phase by 13% and in G2/M phase by 10% when compared with cells transfected with a scrambled siRNA (p<0.05; Figure 4b). Untreated cells showed a reduction in the number of cells in G0/G1 and an increase in the S- and G2/M-phases compared with mock transfected cells, but no statistically significant differences were seen in the cell cycle distribution of cells transfected with scrambled siRNA and mock transfected cells. Ablation of any eEF1B subunit is therefore associated with a small but significant decrease in the proportion of cells in S and G2/M phases and an increase of cells in G0/G1 phase.
In HCT116 cells, down-regulation of eEF1B subunits increased the proportion of cells in G0/G1 by 6%, and reduced the proportion of cells in S-phase by 10% and in G2/M phase by 5% in comparison to cells with scrambled siRNA (p<0.05; Figure 4b). Similarly, in DLD1 cells, knocking down eEF1B subunits increased by 8% the proportion of cells in G0/G1 compared with cells transfected with non-targeting siRNA (p<0.05). Cells in S-phase decreased by over 15% (p<0.01) and cells in G2/M phase decreased by more than 9% (p<0.05; Figure 4b). In all cell lines studied therefore, ablation of eEF1B is associated with a small but significant decrease in the proportion of cells in S and G2/M phases and an increase of cells in G0/G1 phase.
eEF1B subunits co-localise with eEF1A2 using PLA.
eEF1B subunits were found not to bind to eEF1A2 in a yeast 2 hybrid experiment [10]. This was unexpected given the position of the eEF1B binding site relative to amino acid differences between eEF1A1 and eEF1A2 [20]. We therefore sought to look in mammalian cells for evidence of interaction with eEF1A2.
Initially, we used immunofluorescence on sections of mouse spinal cord to see if there was any evidence for co-localisation of eEF1A2 with eEF1Bα and eEF1Bδ (Figure 5). It can be seen that there is strong co-localisation of both eEF1B subunits with eEF1A2 in the neurons (we were unable to test eEF1Bγ because of issues with antibody specificity under these conditions). Both eEF1B subunits also showed expression in non-neuronal cells that do not express eEF1A2.
IF images of the expression of eEF1A2 and eEF1Bα (top panel) or eEF1Bδ (bottom panel) on mouse spinal cord.
In order to obtain more direct evidence for binding, we used the proximity ligation assay (PLA). PLA is based on dual binding by a pair of probes to the two proteins of interest via two specific antibodies raised in different species, in order to generate DNA strands, which then are amplified and serve as surrogate markers for the detected protein molecules [24],[25]. PLA has the advantage of investigating endogenous protein interactions in situ, either in tissues or cultured cells directly.
Figure 6A shows PLA performed in HeLa cells, which express both eEF1A1 and eEF1A2 [26]. Although in one report the longer form of eEF1Bδ was found in HeLa cells [11], the cells we used expressed only shorter eEF1Bδ isoforms (Figure 1A). In HeLa cells eEF1A2 gave positive PLA signals with all eEF1B subunits (Figure 6IC,D and E). PLA using no antibody (5IA), eEF1A2 antibody only (6IB) and eEF1A2 and TK1 antibodies (6IF, a further negative control) gave neither PLA signals nor any background, demonstrating that the signals produced by the eEF1A2/eEF1Bα, eEF1A2/eEF1Bδ and eEF1A2/eEF1Bγ antibody pairs are genuine and specific, and that eEF1A2 does co-localise with eEF1Bα and eEF1Bδ in HeLa cells at the resolution that can be detected by PLA.
Panel I: PLA on HeLa cells. A. Negative control with both primary antibodies omitted. B. eEF1A2 antibody only. C. PLA of eEF1A2 and eEF1Bα. D. PLA of eEF1A2 and eEF1Bδ. E. PLA of eEF1A2 and eEF1Bγ. F. PLA of eEF1A2 and TK1 as negative control. Images in the squares are higher magnification of selected areas. Panel II: PLA on NIH-3T3 cells stably expressing V5-tagged eEF1A1. A. Negative control with both primary antibodies omitted. B. V5 antibody only. C. PLA of eEF1Bα and V5. D. PLA of eEF1Bα and TK1. E. PLA of eEF1Bδ and V5. F. PLA of eEF1Bδ and TK1. G. PLA of eEF1Bγ and V5. H. PLA of eEF1Bγ and TK1. Images in the squares are higher magnification of selected areas. Panel III: PLA on NIH-3T3 cells stably expressing V5-tagged eEF1A2. A. Negative control with both primary antibodies omitted. B. V5 antibody only. C. PLA of eEF1Bα and V5. D. PLA of eEF1Bα and TK1. E. PLA of eEF1Bδ and V5. F. PLA of eEF1Bδ and TK1. G. PLA of eEF1Bγ and V5. H. PLA of eEF1Bγ and TK1. Images in the squares are higher magnification of selected areas.
In order to confirm these results and to show that under these conditions eEF1B subunits also interacted with eEF1A1 (as predicted from early eEF1 complex purification experiments) we used cell lines which stably express either eEF1A1 with a V5 tag or eEF1A2 with a V5 tag (J. Janikiewicz, unpublished). Our antibodies raised against eEF1A1 are not specific under these conditions, and also recognise eEF1A2, so the use of these cell lines permitted us unambiguously to distinguish between the two isoforms.
In V5-eEF1A1 expressing NIH-3T3 cells, a V5 antibody gave positive PLA signals with all eEF1B subunits as expected (Figure 6II). The signals for V5/eEF1Bγ (Figure 6IIG) were not as numerous as those for V5/eEF1Bα or V5/eEF1Bδ (Figure 6IIC and E), but were nevertheless considerably more numerous than those in any of the negative controls. In V5-eEF1A2 expressing NIH-3T3 cells similar results were observed (Figure 6III). The PLA signals were slightly less strong than in V5-eEF1A1 transgenic cells, but the combined results of this analysis and of endogenous eEF1A2 in HeLa cells strongly suggest that eEF1A2 is able to bind to a complex containing all three eEF1B subunits.
We have shown that although each of the components of the eEF1B complex is widely expressed, their expression is not uniform in either tissues or cell lines. eEF1Bδ, in particular, shows an additional layer of complication with alternative splicing and the use of a long additional 5′ exon that gives rise to a much larger protein isoform whose expression is confined to brain (this study, [11]) and testis [11]. In this case, the long isoform has a different subcellular location and function, as it localised to the nucleus where it is involved in induction of the heat shock response. With this exception, all the subunits do, however, appear to be ubiquitously expressed in cell lines and tissues. Both eEF1Bδ and, to a lesser extent, eEF1Bγ, are strongly expressed at E15.5 in both brain and liver, with expression being downregulated by p10. Interestingly, eEF1Bα and eEF1Bδ show an almost reciprocal expression trend during development, with eEF1Bα being expressed more strongly postnatally in both brain and liver, and eEF1Bδ being extremely highly expressed in both tissues at E15.5 and P1 but then dropping markedly by P10. In adult mice, pancreas and liver show consistently high levels of all subunits (Figure 1). Immunohistochemistry shows that the staining in pancreas is particularly intense in pancreatic islets, although this is less marked with the human sample tested (Figure 2). It is impossible to draw solid conclusions from a single sample, though. Both eEF1Bα and eEF1Bδ are strongly expressed in neurons, more so than in glia, and again this is seen in both mouse and human samples.
All the subunits proved eminently amenable to being knocked down using RNA interference (Figure 3), being reduced to near undetectable levels in HeLa cells at 72 hours; similar results were obtained with two other cell lines. Subsequent experiments showed that knocking down eEF1Bγ also affected expression of the other two subunits, so the effects of ablating this subunit cannot be judged in isolation. Knocking down expression of any one of the subunits, in each cell line tested, had a small but significant effect on cell viability, and concomitant modest alteration in cell cycle profile with a reduction of cells in S phase. Surprisingly, knocking down eEF1Bγ seemed to have the lowest effect on viability in two of the cell lines, even though knocking down this subunit also affects expression of eEF1Bα and eEF1Bδ. The difference, if real, might be attributable to the effects on viability being mediated by an imbalance in the subunits rather than a general decrease in expression of the whole complex. In contrast, eEF1Bα has been shown to be essential for growth in yeast [4]. It is possible that this would also be true of mammalian cells over an extended period of time, but certainly our results suggest that they are able to survive and grow over several days with only very low levels of eEF1B. This could imply that mammalian eEF1A isoforms are less dependent on eEF1B for their function in protein synthesis; yeast do not have eEF1A2, and it would be formally possible that the presence of eEF1A2 rescues the loss of eEF1B. However, the biochemical analysis of purified eEF1A2 would suggest that if anything it would be more dependent on GTP exchange than would eEF1A1 [21]. Moreover, the DLD1 cell line was selected for this experiment because, unlike HeLa and HCT116 cells, it does not express eEF1A2 (unpublished observations). The viability of this cell line was slightly lower after ablation of eEF1B than the other cell lines, but not to an extent that seems likely to be significant.
As an important part of the cellular machinery that regulates protein translation elongation as well as being involved in other cellular functions, the structure of eEF1H has been broadly studied. eEF1H is composed of eEF1A and eEF1B, and several models have been proposed for relative placement of each component. However, as there is much inconsistency among these models, further studies were required, particularly in regard to possible differential binding of eEF1A1 and eEF1A2 to eEF1B. In particular, although we showed that regions containing sequence differences between eEF1A1 and eEF1A2 proteins do not harbour eEF1B binding sites, suggesting the two isoforms have similar capability of binding to eEF1B [20], yeast 2 hybrid experiments had previously demonstrated interactions between eEF1A1 and eEF1B, but no interactions between eEF1A2 and eEF1B subunits. This study suggested that eEF1A2 might use a different GTP exchange factor [10]. We therefore used the in situ Proximity Ligation Assay (PLA) technique to attempt to resolve this discrepancy. PLA has been proved to be an efficient and straightforward method to examine endogenous protein-protein interactions in situ, avoiding the possible artefacts from experimenting with isolated or exogenous proteins.
As a first step, we carried out co-localisation analysis of eEF1A2 with eEF1Bα and eEF1Bδ on spinal cord sections and were able to demonstrate clear co-localisation in spinal cord motor neurons. PLA, however, gave false positive signals when used in fixed spinal cord in our hands, so we turned to cell lines. These false positives have also been reported in a study of fixed but not fresh mouse brain [27], possibly due to the deleterious effect of aldehydes on DNA that might benefit the binding of oligonucleotides to the probes during the PLA reaction [27]. These false positives were not observed using cultured cells fixed with the same methods, suggesting that neurons may be more sensitive to the effects of aldehydes on DNA.
PLA was then carried out using HeLa cells, which strongly express both eEF1A1 and eEF1A2. A clear PLA signal was obtained for each of the eEF1B subunits when used with an antibody against native eEF1A2; controls using no antibodies or an antibody to TK1 were all negative.
Since no specific anti-eEF1A1 antibody was available, the PLA of eEF1A1 with eEF1B was carried out on NIH-3T3 cell lines that stably expressed either V5-tagged eEF1A1 or V5-tagged eEF1A2. In both V5-tagged 1A1-3T3 cells and 1A2-3T3 cells, PLA of V5 and all three eEF1B subunits gave positive PLA signals whilst the negative controls were all weak or completely clear. As comparable results were obtained for both eEF1A isoforms, both eEF1A1 and eEF1A2 appear to bind to eEF1B. It seems possible, therefore, that the fusion protein expressed in yeast cells may have failed to keep the native conformation and consequently influenced protein interactions in the Y2H experiments [10]. It is also conceivable that differences in the ability of each isoform to self-associate could have affected the results [33]. With the same Y2H system eEF1A2 in fusion with GAL4 DNA-binding domain was competent in interaction with other eEF1A-binding proteins identified in an Y2H screening [10],[28], but perhaps the fusion protein was modified in a way that masked the binding sites for eEF1B.
The other major difference between the result in our study and the Y2H experiments is that in transgenic NIH-3T3 cells we also detected an interaction between V5-tagged eEF1A1 and eEF1Bγ, which was not found in the Y2H experiments. This is presumably because that eEF1A and eEF1Bγ are not directly binding each other, but form a complex bridged by another eEF1B subunit, as suggested in most of the eEF1H structure models proposed so far. In PLA the maximum distance between the two probes that allows DNA hybridization and thus PLA signal is around 16 nm [27]. Including the two primary antibodies and the two probes, the distance for two proteins to be recognized as being in proximity by PLA is estimated at roughly 30–50 nm, depending on the sizes of the antibodies used [29],[30]. The limitation of the PLA technique in terms of distinguishing between binding to specific subunits rather than proximity due to binding of the test protein to an entire complex means that we can not deduce unequivocally whether there are differences in the binding of eEF1A1 and eEF1A2 to specific subunits. We can, however, say that both eEF1A1 and eEF1A2 bind to the eEF1B complex.
In conclusion, we suggest that both eEF1A isoforms can bind to eEF1B subunits in vivo. This does not preclude the possibility of a further GTP exchange factor binding to eEF1A2, but this result coupled with our previous comparative homology modelling makes it likely that eEF1A2 does indeed use eEF1B as a GTP exchange factor. Furthermore, the STRING database of protein-protein interactions version 9.1 shows evidence for binding of eEF1Bα and eEF1Bδ to eEF1A2 in human cells, and of binding of eEF1Bδ and eEF1Bγ to eEF1A2 in mouse cells. Again, it is likely that these results indicate binding of the eEF1B complex to eEF1A2 rather than telling us about binding of individual subunits, as all available biochemical evidence suggests strongly that the gamma subunit does not bind directly to eEF1A. Mammalian cells appear to be able to survive for at least periods of a few days in culture in the absence of any given eEF1B subunit, but of course the absence of the whole complex could be lethal.
The recent discovery of missense mutations in eEF1A2 that cause epilepsy, severe intellectual disability and autism is likely to cause more focus on the role of eEF1B binding, as the G70S mutation abuts the eEF1B binding site[34],[35]. It is also of note that a homozygous splice site mutation in the gene encoding eEF1Bα has been found in a family with recessive intellectual disability, suggesting that translation elongation factors may have an important neurodevelopmental role to play in cognition [31]. Further studies on the role of eEF1B in neurons is now a priority.
Mice were housed in the Biomedical Research Facility (BRF) at the University of Edinburgh. All mice were maintained in accordance with Home Office regulations and all protocols had been approved by the local ethics committee of the University of Edinburgh. Wasted mice were closely observed for overall clinical condition and were euthanized where necessary to avoid suffering.
Cell culture
HeLa, HCT116 and DLD1 cells were all obtained locally and cultured according to ATCC guidelines.
Western blotting
Westerns were carried out using antibodies and techniques as previously described [32], with the following antibodies: anti-eEF1Bα from Proteintech (Manchester, UK; 1/2000) or Abcam (Cambridge, UK; 1/400), anti-eEF1Bδ from Proteintech Group (Manchester, UK; 1/3000), anti-eEF1Bγ from Abnova (Taipei, Taiwan; 1/2000) or Abcam (Cambridge, UK; 1/1000) and anti-GAPDH from Chemicon (Merck Millipore, Watford, UK; 1∶30,000). Calbiochem (Merck Millipore, Watford, UK) Rapid Step ECL was used for detection.
Slides of human tissues were obtained from Biochain (AMS Biotechnology, Abingdon, UK). Paraffin embedded sections of human and mouse tissues were deparaffinised, blocked in peroxidise blocking solution for 5 minutes and then washed and blocked in goat serum diluted 1∶5 with PBS for 10 minutes. Primary antibody was added as follows: anti-eEF1Bα from Proteintech (Manchester, UK; 1/100), anti-eEF1Bδ from Proteintech Group (Manchester, UK; 1/400) and anti-eEF1Bγ from Abnova (Taipei, Taiwan; 1/100). The slides were then incubated and visualised using ChemMate DAKO EnVision Detection Kit (DAKO) according to the manufacturer's instructions. In brief, the slides were then washed in PBS and three drops of ChemMate DAKO Envision/HRP Rabbit/Mouse secondary antibody (DAKO Cytomation; Agilent Technologies, Wokingham, UK) were added to each slide and incubated for a further 30 minutes. The slides were washed with PBS, removed from the sequenzer and 0.5 ml of DAB working solution was added to each slide and incubated for 2 minutes. Finally, the slides were washed in dH2O, counterstained in haemotoxylin, stained with lithium carbonate and dehydrated in absolute ethanol and 75% ethanol, cleared in xylene and mounted in pertex. The entire procedure was performed at room temperature. Sections were viewed by light microscopy on Olympus BX51 using DP software (Olympus).
RNA interference
Silencer siRNAs were obtained from Ambion (Paisley, UK), and experiments were controlled using untransfected cells and cells transfected with a scrambled siRNA. Two siRNAs and one Silencer Select were obtained for each eEF1B subunits. Each of the siRNAs was resuspended to a concentration of 100 µM and stored at −70°C. Transfections were performed using a Nucleofector and kits supplied by Amaxa. For each well on a 6-well plate, 0.5×106 cells were pelleted, resuspended in the appropriate volume of siRNAs and 100 µl of nucleofector solution (Amaxa Biosystems, now Lonza). Cells were then subjected to nucleofection a. HeLa cells were transfected with 30 nM of either a siRNA oligonucleotide targeting a particular eEF1B subunit or a non-targeting control by nucleofection. Cells were harvested 72 h after transfection and analysed by Western blot as above.
Cell cycle analysis
For cell cycle analysis, cells were resuspended and incubated in 500 µl propidium iodide staining solution for 20 minutes before being analysed using a Coulter EPICS XL flow cytometer (Beckman Coulter). A dot-plot was drawn of forward light scatter (FLS) against side scatter (SSC), which are influenced by size and refractive index, and all cells were gated for further analysis except dead cells and cell debris by using the EXPO ADC analysis software. A FL3 histogram with a linear x axis was used to visualise the DNA content of the cells. FL3 histogram was obtained from 10,000 events data. Multicycle AV software (Phoenix Flow systems) was used to analyse the output. Statistical comparisons were carried out by estimating the standard error of the mean and testing for significant differences from the results obtained with the non-targeting siRNA using Student's t-test.
Cell proliferation assay
Alamar blue (Life Technologies, Paisley, UK) assays were carried out according to the manufacturer's instructions and the signal was read by fluorometry (Biotech synergy HT Plate Reader - Fisher Scientific). The fluorescence readings were taken at excitation of 560 nm and emission of 590 nm.
Slides were blocked for 30 minutes with donkey diluted 1∶10 in PBS. Slides were incubated with primary antibodies for 10 minutes then HRP conjugated rabbit antibody 594 and HRP conjugated sheep antibody 488, red and green respectively, were added to the slides, and the slides were incubated in the dark at room temperature for 30 minutes before being washed in PBS for 5 minutes. The slides were sealed with coverslips using Vectashield (Vector Laboratories, Peterborough, UK) hard set mounting medium with DAPI and observed under a Zeiss Axioskop 2 fluorescence microscope using appropriate filters, and pictures were captured using Smart Capture 2 software.
Proximity Ligation Assay
PLA was performed using reagents and directions supplied in the Duolink in situ PLA kit, All the incubation processes were performed in a pre-warmed humidity chamber at 37°C. Negative controls included a sample with only one primary antibody to one of the target proteins, and a sample with a pair of primary antibodies raised against one target protein, and a protein that was not expected to interact with the target protein (based on function and/or subcellular localisation; TK1, antibody included with kit).
We are grateful to Dinesh Soares and Helen Newbery for their help with this project.
Author Contributions
Conceived and designed the experiments: YC MP JJ JD CA. Performed the experiments: YC MP JD. Analyzed the data: YC MP CA. Contributed reagents/materials/analysis tools: JJ. Wrote the paper: YC MP CA.
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Aveeno Skin Relief Lotion 200ml
£5.00£2.50 per 100ml
Product information
AVEENO® Skin Relief Moisturising Lotion, with a rich, fast absorbing and non-greasy formula. Clinically proven to moisture and effectively soothe very dry, irritable or sensitive skin. Even suitable for diabetic's rough, dry skin. Formulated with pre-biotic triple oat complex* (oat flour + oat extract + oat oil), it helps to improve the balance of the skin's natural microbiome. Leaving skin more resilient and healthy looking from day 1.
*In vitro test
AVEENO® harnesses the goodness of nature and the power of science to keep your skin looking healthy and feeling balanced.
Country of Origin
Ratings and reviews
Customer reviews (7)
The Best
Quite simply the best cream I've ever used, and I've tried a lot over the years. I suffer from numerous skin conditions and this is the only cream that seems to combat all of them. I can't recommend this product enough.
magic cream
I've tried all types of cream for my dry skin & eczema patches and nothing works. this cream has not only helped relieve the itchy patches, calmed the redness but they have all gone. I cannot rave on enough about this cream. one down side is the cost it's cheaper in other places
This cream is so good at moisturising after application my skin feels soft and comfortable. It stays on for ages even when getting your hands wet. It plumps your skin up too!!
Feels great on skin. Not greasy. i bought it to see if it helps with my Dermatitis flare ups on my face. ive read on google this can help so time wil tell
Just as described
This is amazing on your skin and absorbs really quickly.
Great Product
The only cream that will get rid of my painful eczema
of 2


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The cables solve the purpose of underwater working. The cable manufacturers have to follow the IEC standard 60288 in order to make them stable against marine animals, tsunamis or by fishermen trawls.
- These wires can withstand any temperature on land as well as seabed
- Submarine wires should have no effect on the depth of sea or ocean
- For its use in deep water, the submarine cables compile with the special protection layer.
- The submarine cables are highly thermal resistive of seabed and land
Also Read: Submarine – principles and design? How does it work?
Joints and laying procedure of Submarine cables:
The testing of the cable joint is necessary before executing them under the high pressure of the sea. The joints have to pass all the insulation, dielectric and radiographic test before using the cable which gets too old or damaged.
The procedure of laying the Submarine cables is a tough laborious job and it depends on different parameters of the project. But still it can be done with the help of following steps:
- There are preliminary checks of cables on various stages while loading.
- Putting the first end of the submarine cable in the sea or ocean will be the next step after all checks.
- The next steps are to laying a cable between two terminals i.e. on and under the seabed
- At the end land the other terminal of the cable.


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Tennis Channel Asks FCC to Force Comcast Compliance With ALJ Ruling
Tennis Channel has asked the Federal Communications Commission to force Comcast to comply with the terms of the Administrative Law Judge ruling that the MSO violated the program carriage rules by favoring its own similarly situated networks, Golf Channel and Versus, now known as the NBC Sports Network.
"The Chief Judge ordered Comcast to remedy its discrimination by providing equal carriage treatment
'as soon as practicable," wrote Tennis Channel in its complaint, according to a copy supplied to Multichannel News.
In the complaint, Tennis said the language is clear that such a ruling becomes effective upon release, though it is subject to appeal to the commission, which Comcast has done.
The ALJ decision also says the ruling becomes effective unless "exceptions" to it are lodged, which Comcast has done in spades, even asking for a waiver of the 25-page limit for such appeals to it can add another 15 pages worth of exceptions.
"The factual and legal issues presented in the Initial Decision are complex and raise serious questions before the Commission, including but not limited to whether the Initial Decision is consistent with Commission precedent, and whether its unprecedented mandatory carriage remedy violates the First Amendment," Comcast said in its motion for more pages.
But Tennis, which has been returned to Verizon FiOS systems through a new carriage agreement with the telco, says it would be hurt by delayed implementation.
"It would punish Tennis Channel and reward Comcast for its discrimination by allowing Comcast to avoid compliance even after it has been found to have violated Section 616-a violation found by the ALl to be sufficiently egregious to warrant the imposition of the largest forfeiture available to the Commission under the law," said the channel in its filing.
That payment is $375,000.


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Did your last computer break? Have you been looking for something constrictive and environmentally-friendly to do with the leftover parts? If so, then you may want to consider recycling those parts, or in this case, upcycling them. Upcycling is the process of creating a brand new product out of parts of old products, and it is growing in popularity. When you upcycle a product, you reduce the amount of parts you have to throw away, and thus, you also reduce the impact you will make on the environment. If you are looking for fun ways to reduce your impact, reuse old computer parts, and recycle them into something new, then consider one of the following ideas:
A Computer Shell Bookcase
If you have a desktop computer that you are trying to recycle, consider making a bookcase out of the exterior shell. You can attach some wood to the bottom to help the shell stand upright, and then you can put wood at the top and on the back to enclose the books inside the case. Then you can just start putting books next to each other inside of this little bookcase you've made. You may only be able to fit three to five books at a time in there, but if you have multiple computers, you can repeat the above process, attaching the bottom of one "shelf" to the top of the next one until you have a towering bookcase that stands several feet tall and can accommodate books of various sizes.
Hard Drive Coaster
For the overly ambitious person, one of the best ways to upcycle old computer parts may be to turn the hard drive into a coaster. Once you pull the hard drive out of your computer, you can then take a piece of steel or aluminum and weld it over the top of the hard drive, rendering it all one flat, closed piece. Then, you can just set this on a table somewhere and set your drink glass on it if you need a place to set your beverage without staining the table.
LCD Monitor Mirror
You can be classy, stylish, and environmentally-friendly by turning your old LCD monitor into a mirror. Using the utmost care, remove the LCD panel and all of the internal parts from the outer casing of your monitor. Dispose of the LCD panel immediately, as there is really no safe way for you to reuse this. Then, get a glass panel that is cut to the same size as the interior of the monitor casing or the same size as the LCD panel. Insert this glass into the monitor casing, then reattach the back of the monitor to the casing. Make sure all of the screws and connections are tightened, and make any reinforcements as necessary. Then, put the monitor back on its stand and place it on a dresser or table where you will see it often and can use it as a mirror.
If you've been looking for responsible ways of recycling your old computer parts without having to throw them all out and without harming the environment, then consider making one of the three projects above. Not only will you be acting friendly towards the earth, but you will also end up with some pretty cool items that you may not otherwise have had. Contact a company like Computer Backup Inc for more information.Share


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Enchanted is a video game for the Nintendo DS based on the Walt Disney Pictures' 2007 film of the same title. In the animated fantasy kingdom of Andalasia, Giselle and Prince Edward are planning to get married. When Edward's evil stepmother tries to stop the marriage, Giselle is transported into New York City. Edward, along with Giselle's chipmunk, Pip, must rescue Giselle from the real world, while she meddles into the lives of an attorney, Robert, and his daughter, Morgan.
The player adventures through Andalasia and New York City as Giselle, Prince Edward, and Pip, each with their own style of gameplay.
A video game is an electronic game that involves human interaction with a user interface to generate visual feedback on a video device such as a TV screen or computer monitor. The word video in video game traditionally referred to a raster display device, but it now implies any type of display device that can produce two- or three-dimensional images. Video games are sometimes believed to be a form of art, but this designation is controversial.
The electronic systems used to play video games are known as platforms; examples of these are personal computers and video game consoles. These platforms range from large mainframe computers to small handheld computing devices. Specialized video games such as arcade games, while common in the 1980s, have gradually declined in use due to the widespread availability of home video game devices (e.g., PlayStation 4 and Xbox One) and video games on desktop and laptop computers and smartphones.
The input device used for games, the game controller, varies across platforms. Common controllers include gamepads, mouses, keyboards, joysticks, the touchscreens of mobile devices and buttons. In addition to video and (in most cases) audio feedback, some games include haptic, vibration or force feedback peripherals.
Marvel 2099: One Nation Under Doom was a cancelled game for the PlayStation developed by Mindscape Inc. It was to be loosely based on the "One Nation Under Doom" storyline in Marvel's Doom 2099 comic. It was envisioned as a 2D side-scroller with 3D rendered characters.
In February 1996, Mindscape announced they would produce a video game based on the Marvel 2099 universe. Promoted as one of their top five games for the year, the game would be released on December 1, 1996, just in time for the Christmas rush. Two versions were announced: PlayStation and Windows 95.
By May 1996, CD-ROM and VHS video demos were being shipped to game magazines for pre-release reviews, along with a one-page color brochure. The first public demo was shown at the E3 show, and featured a playable single level of the Punisher 2099 fighting SHIELD troops, and also opening menus and some cut scenes. Electronic Gaming Monthly had a quarter-page preview of the game in their July 1996 issue and a half page preview in their August 1996 issue, showing screenshots of actual gameplay, and a group shot of the player characters. At the 1996 San Diego Comicon, the Mindscape booth handed out brochures, and raffled off One Nation Under Doom pins, shirts, and posters. Some attendees were even allowed to play the demo at the booth, although no copies of the demo were distributed. September 1996 issue of 3D Design magazine had a cover story on the Marvel 2099 game. After July, due to financial troubles, ongoing production of the game slowed down, and eventually stopped, though the game was never officially canceled.
Mario Party (Japanese: マリオパーティ, Hepburn: Mario Pāti) is a party video game series featuring Mario franchise characters in which four human- or computer-controlled characters compete in a board game interspersed with minigames. The series was developed by Hudson Soft and published by Nintendo; the arcade version was developed by Capcom. The series is known for its party game elements, including the often-unpredictable multiplayer modes that allow play with up to four (and sometimes eight) human players.
After the development of Mario Party 8, several of Hudson Soft's key designers left to work for Nintendo subsidiary Nd Cube, developers of Wii Party. Starting in 2012 with Mario Party 9, Nd Cube has taken over development of the series from Hudson Soft. The latest title in the series, Mario Party 10 was released worldwide in March 2015 on Wii U.
The series currently holds the record for the longest-running minigame series. As of December 2014, Nintendo reported cumulative worldwide sales of 39.6 million game copies in the Mario Party franchise.
Enchanted (or The Enchanted Works of Stevie Nicks) is a three disc box set of material by American singer-songwriter and Fleetwood Mac vocalist Stevie Nicks, which encompasses her solo career from 1981's Bella Donna to 1994's Street Angel.
The box set also contains rare B-sides, unreleased live recordings, demos, songs from soundtracks, a track from the 1973 Buckingham Nicks album, outtakes from the Bella Donna and Rock a Little sessions, and a new recording of the classic song, Rhiannon. Some previously released tracks are also in noticeably longer versions, or alternative remixes.
The set was released on April 28, 1998 and peaked at #85 on the Billboard 200 Albums chart. Enchanted was certified Gold by the RIAA on April 7, 1999 For Shipments of over 500,000 copies. Two singles were released, "Reconsider Me" (a deleted track from the 1985 Rock a Little album) and "Enchanted" which made its first appearance on 1983's The Wild Heart album.
It was mastered using HDCD technology and, although it is unmarked, playing the album in a CD player able to decode HDCD will give superior sound quality.
An immersive video is basically a video recording of a real world scene, where the view in every direction is recorded at the same time. During playback the viewer has control of the viewing direction. Generally the only area that can't be viewed is the view toward the camera support. The material is recorded as data which when played back through a software player allows the user control of the viewing direction and playback speed. The player control is typically via a mouse or other sensing device and the playback view is typically 4:3 window on a computer display or projection screen or other presentation device such as a head mounted display.
A recent example, as of 2011, of immersive video is provided by the Kogeto Dot panoramic video capture device and associated online community for smartphones, for the iPhone 4, iPhone 4S and for the Galaxy Nexus with the Kogeto Dot 360-degree panoramic video accessory.
Sometimes the phrase "immersive video" is used to describe full-size teleconferencing capabilities, such as Cisco's Telepresence product.
Microsoft Video 1 or MS-CRAM is an early lossy video compression and decompression algorithm (codec) that was released with version 1.0 of Microsoft's Video for Windows in November 1992. It is based on MotiVE, a vector quantization codec which Microsoft licensed from Media Vision. In 1993, Media Vision marketed the Pro Movie Spectrum, an ISA board that captured video in both raw and MSV1 formats (the MSV1 processing was done in hardware on the board).
Microsoft Video 1 operates either in a 8-bit palettized color space or in a 15-bit RGB color space. Each frame is split into 4×4 pixel blocks. Each 4×4 pixel block can be coded in one of three modes: skip, 2-color or 8-color. In skip mode, the content from the previous frame is copied to the current frame in a conditional replenishment fashion. In 2-color mode, two colors per 4×4 block are transmitted, and 1 bit per pixel is used to select between the two colors. In 8-color mode, the same scheme applies with 2 colors per 2×2 block. This can be interpreted as a 2-color palette which is locally adapted on either a 4×4 block basis or a 2×2 block basis. Interpreted as vector quantization, vectors with components red, green, and blue are quantized using a forward adaptive codebook with two entries.


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is made on-demand. We will ship it separately in 10 to 15 days.
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© 2020 EMSÉ studio


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