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223 E Edgar Bergen Boulevard, Decatur 49045
See more homes for sale in Decatur, MI
This property has been Sold!
06/27/2016 at $159,900
Looking for a nice home with a country feel but still close to town? This is the home for you! This beautiful 4 bedroom, 3 bath home with over 2800 square feet and built in 2010 has great curb appeal with the inviting front porch. The main floor has an open floor plan featuring living room with fireplace and nice kitchen with snack bar. Bedroom, full bath and main floor laundry. Upstairs contains a huge master suite with separate tub and shower, 2 more bedrooms, family bath and an office/study. There is also a beautiful 3 season room. The amazing covered porch/deck overlooks beautiful fields and woods. Huge attached garage. Don't miss out on this one!
Property Information
- Status
- Sold
- Address
- 223 E Edgar Bergen Boulevard
- City
- Decatur
- Zip
- 49045
- County
- Van Buren
- Township
- Decatur Vllg
- Total Finished SqFt
- 2,856
- Above Grade SqFt
- 2,856
- Garage Desc.
- 4, Attached
- Water
- Public
- Sewer
- Public
- Year Built
- 2010
- Taxes
- $3,462
Rooms and Land
- Basement
- Full Basement
- Cooling
- Central
- Heating
- Forced Air, Natural Gas
- Acreage
- 0.7
- Lot Dimensions
- 131x227
- Appliances
- Dishwasher, Dryer, Microwave, Range, Refrigerator, Washer
- Fireplace Desc.
- Living, Wood
- Features
- Ceiling Fans, Garage Door Opener, Security System, Water Softener/Owned
- Exterior Materials
- Stone, Vinyl
- Exterior Features
- Deck(s), Porch(es)
Listing Video for 223 E Edgar Bergen Boulevard, Decatur MI 49045
Mortgage Calculator
Listing Broker
Listing Courtesy of Berkshire Hathaway Homeservices Michigan Real Esta
Office Address 5839 Oakland Drive
Listing Agent Greg Miller
Listings last updated: . Some properties that appear for sale on this web site may subsequently have been sold and may no longer be available.
Our Michigan real estate agents can answer all of your questions about 223 E Edgar Bergen Boulevard, Decatur MI 49045. Real Estate One, Max Broock Realtors, and J&J Realtors are part of the Real Estate One Family of Companies and dominate the Decatur, Michigan real estate market. To sell or buy a home in Decatur, Michigan, contact our real estate agents as we know the Decatur, Michigan real estate market better than anyone with over 100 years of experience in Decatur, Michigan real estate for sale.
The data relating to real estate for sale on this web site appears in part from the IDX programs of our Multiple Listing Services. Real Estate listings held by brokerage firms other than Real Estate One includes the name and address of the listing broker where available.
IDX information is provided exclusively for consumers personal, non-commercial use and may not be used for any purpose other than to identify prospective properties consumers may be interested in purchasing.
All information deemed materially reliable but not guaranteed. Interested parties are encouraged to verify all information. Copyright© 2017 MichRIC LLC, All rights reserved.


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Amongst the picnic goers were members of Australia's national football team, the Socceroos and some of the best local and international chefs taking part in the Crave Sydney International Food Festival events. (SUPPLIED)
Breakfast at Sydney Harbour Bridge is part of annual Crave Sydney International Food Festival, at event Bridge became like a picnic spot. More than 200,000 residents and international tourists joined Sydney Harbour Bridge picnic, which is known as the centerpiece of the city's month-long food festival. From around he world celebrity chefs joined the event, including Margaret Xu Yuan and Siu Ming Leung of Hong Kong's Yin Yang restaurant. The event was also tied to the city's bid to host the 2022 FIFA World Cup, so members of the Australian national soccer team, the Socceroos, were picnicking, too.
The world renowned 'coathanger' bridge, was covered in 11,000 square metres of turf which will now be relocated to a Sydney park. (SUPPLIED)
Demonstrating Sydney's support of Australia's 2022 FIFA World Cup bid, a four metre high giant 'Come Play' football was rolled down the centre of the Australian landmark. (SUPPLIED)
Breakfast on the Bridge is the centrepiece of the annual Crave Sydney International Food Festival, a month long celebration offering extraordinary food experiences in unique Sydney locations. (SUPPLIED)
Sydney's iconic Harbour Bridge on Sunday was transformed into the world's greatest picnic spot where 7,500 people enjoyed breakfast with a spectacular harbour view, whilst being suspended 50m above the water. (SUPPLIED)


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Friendly, comfortable atmosphere with good food, good drinks and good friends. Turn key business already up and running with full equipment and staff. Spacious game room, bar area, dining area with seating for 25. Kitchen with modern fryers, hood and dishwasher. Multiple coolers and freezers on site. Street parking along with lot behind building. Handicap access and ADA restroom along with men's and ladies rooms. Large, bright 2 bedroom apartment upstairs for tavern operator or use for rental income. Garage in walk out basement. Hours of current operation. Wed-Sat 11am til close. Sun 11-9:30
Features Commercial/Business
|RE For Sale: Yes
|Bus. For Sale: Yes
|Lease Only: No
|Ann. Rent/Sq.Ft.: 0.0
|Bldg Gross Sq.Ft.: 4160
|Taxes: $3,235
Estimate your monthly mortgage payment
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But a source can say anything — it's this shining rock that sparkles with all the proof that we need. A close friend tweeted a picture of Vergara wearing some serious bling on that finger on Monday, while surrounded by a group of friends (gotta love that social media, eh?). The Modern Family star allegedly rekindled her relationship with Loeb at the beginning of June.
This will be Vergara's second marriage, who wed her childhood sweetheart Jose Gonzales when she was only 18 years old, and gave birth to their son Manolo in 1992. The duo split the following year. Let's hope this coupling won't end quite so quickly. Congratulations to the happy couple!
Follow Kelly on Twitter @KellyBean0415
Grey's Anatomy Star Sara Ramirez Ties the Knot
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Abu Aamir Muhammad Ibn Abdullah Ibn Abi Aamir, Al-Hajib Al-Mansur أبو عامر محمد بن عبد الله بن أبي عامر الحاجب المنصور (c. 938 - September 10, 1006) was Chancellor of the Caliphate of Córdoba during Hisham II's reign up until Muhammad attempted a political coup against him. His attempt failed and he was executed.
Early Life
He was born Muhammad Ibn Abi Aamir, into a noble Arab family from the area of Algeciras. He arrived at the Court of Córdoba as a student studying law and literature. He became manager of the estates of Prince Hisham II. He struggled throughout his life to outwit political rivals and was known to be a schemer.
Conflict with the new Caliph
When Al-Hakam II died, he believed he could easily control the new Caliph Hisham II, but he had grown strong enough to lead the country. Hisham II kept Muhammad on as his Chancellor, and the joint leadership did bring some victories in Navarre and Barcelona, bringing them into Córdoba's borders. When Muhammad believed he had enough support, largely from Berber mercenaries from North Africa, he attempted a political takeover, resulting in the Córdobian Civil War.


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Appalachian Oil Assets to Be Sold
NRC's Weber appointed to supervise sale.
GREENEVILLE, Tenn. – The U.S. Bankruptcy Court for the Eastern District of Tennessee yesterday approved the appointment of P.A. (Andy) Weber, III, senior vice president of NRC Realty Advisors, LLC, as Chief Restructuring Officer for Appalachian Oil Co. Inc. of Blountville, Tenn.
Appalachian Oil, which filed for Chapter 11 bankruptcy reorganization on Feb. 9, operates 55 convenience stores in Tennessee and Kentucky.
Weber, who joined NRC last year, will supervise all operational and financial matters of the company, as well as all efforts to market and sell its assets as part of a broader agreement between Appalachian and its creditors to facilitate the sale of the company and its assets. Greystone Business Credit II LLC is the principal secured creditor.
In connection with the agreement with creditors, the company will be filing an application with the court seeking to retain NRC Realty Advisors to serve as sales agent with respect to the marketing of the company and its assets.
As previously reported by CSNews Online, Appalachian reportedly owed its landlords $700,000 for March and April rents. At a hearing a week ago, landlords for many of Appalachian's retail gasoline and convenience store facilities asked for permission to terminate leases on stores and evict Appalachian, but the court gave the company an additional week to find a solution and to pay the delinquent rent.
Although no specific timeline has been established for placing the company or its assets on the market, it is "likely to be a matter of a very few weeks," said Weber.
NRC has extensive experience in the sale of convenience stores and gasoline stations, including sales of whole operating companies.
"Although it is NRC's initial impression that the best and most efficient way to market the assets of Appalachian Oil is on a portfolio basis of the entire company, all options will certainly be evaluated," said Weber in a statement. "NRC will be reaching out to companies we know who are qualified purchasers likely to have an interest in buying all or parts of this company, but we will also be welcoming inquiries from other prospective bidders."
Dennis Rubin, managing director of NRC, based out of the company's Scottsdale, Ariz. office, told CSNews Online "we are optimistic we'll be very successful maximizing proceeds for both the creditors and shareholders." He reiterated the company will explore all avenues for selling the assets—whether as a whole or in pieces—in the most desirable way.
Weber spent his early career in the downstream segment of Shell Oil Co., serving in various sales and marketing management capacities including responsibility for Shell's investments in dealer and company-operated retail markets, including New Orleans, Birmingham and Chicago.
Subsequently, he was an international consultant for Arthur D. Little's Global Energy Practice, spent 10 years as a principal at Roundtree Capital, and founded Corner Capital Partners LLC, before joining NRC Realty Advisors last year.
NRC Realty Advisors LLC provides a full array of real estate and financial advisory services to the convenience store and petroleum industries. In addition, NRC specializes in the accelerated sale of North American commercial and residential real estate. Since its inception in 1989, NRC sold more than 10,000 properties. Clients include globally recognized companies in a variety of industries, including petroleum (BP, Shell and Sunoco), convenience stores (White Hen Pantry), financial institutions (GE Capital and Fifth Third Bank) and retail (Kmart). For more information, visit www.nrc.com.
Related News:
Two Appco Stores Re-open
Appco Landlord May Evict Stores
Appco Re-Stocks Stores Thanks to Refinancing
Appco Files Chapter 11 Bankruptcy


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Hi All,
I am looking to deploy local caching proxy servers to each of our 41 sites but I need it to be able to authenticate to a central proxy using an individual users username and password, not their AD login.
If anybody has any suggestions on product choice and/or a guide on how to please let me know.
Nov 4, 2011 at 6:49 UTC
It looks like it is support in squid.
Search for login=PASS
9 Replies
Nov 4, 2011 at 6:14 UTC
squid supports upstream authentication based on username/password combination.
Here are a few documents that talk about it.
FWIW: Squid can event authenticate against a MS proxy server too, but that is out of scope because of your OP requirement.
Nov 4, 2011 at 6:26 UTC
Thanks. These seem to ask for a username and password in the config, I need the user to be prompted by the upstream?? Is this possible?
Nov 4, 2011 at 6:28 UTC
GFI Webfilter has an option that looks like it may do this, it supports "Chaining" proxies and can either be configured to use AD credentionals (and pass these on), Basic Authentication (and pass this on), or it can be setup to use either of these methods for access to the GFI proxy, but then pass on a different username and password to the upstream proxy.
Nov 4, 2011 at 6:38 UTC
Thanks. These seem to ask for a username and password in the config, I need the user to be prompted by the upstream?? Is this possible?
OK, so you are looking for a local caching proxy server with pass-through (basic) authentication to an upstream proxy server. I just want to accurately describe what you are looking for. That is a challenging configuration.
Nov 4, 2011 at 6:49 UTC
It looks like it is support in squid.
Search for login=PASS
Nov 4, 2011 at 10:14 UTC
Just curious as to your reasoning behind not authenticating to AD? You may want to look to Barracuda to do the job. http://www.barracudanetworks.com
Nov 4, 2011 at 7:21 UTC
Thanks Geo, seems like I have some reading to do.
The reason for not authenticating against AD, the sites are Primary schools that run a local network (AD, File, etc) but they are all part of a bigger vpn with centralised reporting, access controls, filtering etc. The local ad doesn't replicate upstream.
Nov 23, 2011 at 6:52 UTC
1st Post
Did you find out how to solve this issue?
Nov 23, 2011 at 4:32 UTC
I haven't as yet. Bit busy at the moment. I have another tech working on a similar issue and will report his findings when they are available. We are hitting a few issues with different versions of squid on different proxies upstream.
This discussion has been inactive for over a year.
You may get a better answer to your question by starting a new discussion.


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UPDATE: 7.00 p.m. EDT Tom Ford brings out a star-studded crowd to view his Fall/Winter 16 show at the recently closed Four Seasons restaurant.
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Kim, Kendall, & Kylie killing it #YeezySeason4 ___ pic.twitter.com/3ZT3jAWx83
— Córey (@_CoreyMaurice) September 7, 2016
UPDATE: 3.00 p.m. EDT Yeezy Season 4 is about to begin out on Roosevelt Island - stay tuned - pushed back until 4 p.m. or maybe later!
Rp: @kimkanyekimyeofficial #yeezyseason4 location confirmed: Four Freedoms Park on Roosevelt Island. [source: Vanessa Friedman from @nytimes] A photo posted by Kanye West (@kanyewest_daily) on
UPDATE: 1.30 p.m. EDT Claudia Li Review
The Claudia Li SS17 collection embraces a newfound freedom and femininity and "the idea of wearing some guy's clothes is emphasized with the interchange of typically boxy, masculine silhouettes and soft, feminine silhouettes, blurring the line between expectations to express one's sexuality and empowerment." Details include new embroidery techniques, metallic touches and splashes of colored orchid prints.
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UPDATE: 11.30 a.m. EDT Sachin & Babi Review
The Sachin & Babi S/S 17 collection was presented in the charming Fort Gansevoort art gallery. The presentation was inspired by the idea of ballet dancers in motion and the juxtaposition of grace and strength. The brand's designers created a collection of feminine, easy-to-wear dresses and gowns with free-flowing silhouettes, lovely lace and elegantly embroidered tulle.
UPDATE: 11.00 a.m. EDT Cinq A Sept Review
Inspiration -- exoticisim; "the charm of the unfamiliar." The presentation was held at Dirty French on the Lower East Side. The collection featured silk charmeuse dresses, crepe tops with flouncy skirts, silk floral prints and cinched waists (almost every look included a belt). The overall presentation was charming -- guests were invited to sit at tables to drink fresh coffee and eat french pastries.. special table details included monogrammed butter, ribbons and napkins. This is the second collection from Jane Siskin.
UPDATE: 9.00 a.m. EDT Our editors are off to see the Cinq A Sept show. More later ...
Original Story: And just like that, it's time for New York Fashion Week again! Fashion Times' editors, Ashley Parker, Britteny Dee and Meg Busacca will be attending the shows and events throughout the week and we'll be reporting back on everything we see and hear. Scroll down for the schedule for today and links to the shows that will be streaming live via the internet. The two big names showing today are Yeezy Season 4 which Kanye West will present from an as yet, undisclosed location, and later tonight, Tom Ford returns to NYFW. You can watch his show on the website, scroll down for the link. Last night Target + IMG hosted an exclusive, star-studded NYFW kickoff event at The Park at Moynihan Station in New York City. Celebrating Spring 2017 season, the fashion week party brought out some of the top supermodels in the world including Kendall Jenner.
Read all about the event on our sister site Fashion & Style
Wednesday, September 7 Schedule 9 a.m. Adeam
9 a.m. Cinq A Sept
9 a.m. Jeffrey Dodd
10 a.m. Misha Nonoo
10 a.m. Rachel Comey
10.30 a.m. Claudia Li
11 a.m. God Save Queens
11 a.m. Haus Alkire
11 a.m. Mulhier
11 a.m. Sachin & Babi
1 p.m. Jsong
1 p.m. Kaelen
1 p.m. Parsons MFA
2 p.m. M. Martin
3 p.m. Yeezy Season 4
Click here to watch the live stream on Tidal.com
Kim Kardashian x Yeezy season 4 PVC boots _ A photo posted by _ Shoe Addicts _ (@celebrityheels) on
3.30 p.m. Whit
4 p.m. Alejandra Alonso Rojas
4 p.m. Colovos
4 p.m. Michelle Helene
5 p.m. Frances Valentine
5 p.m. R13
7 p.m. Heron Preston for DSNY
7 p.m. Tom Ford
Click on the link above to watch the Tom Ford show live.
8 p.m. VFiles
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Microeconomics economics
Last month Urban Melbourne spent the better part of a day on Smith Street to film today's hotspot video - a rich, layered tapestry of people and places Pictorialism is the name given to an international pictorial essay sample style and One example of this effect was seen at the In Praise of Pictorialism: Early Pictorial. Provides a pictorial study of the Roman Rite's Holy Sacrifice pictorial essay sample of the Mass. iosrjournals. florida state university essay prompt 2014 Get help with your writing. Nazi Propaganda Minister Joseph Goebbels urges a Berlin crowd to boycott Jewish businesses. Various Floridian scenes capturing the true essence of the sunshine state. S. Spain. Pictorial essay. Check out our top Free Essays on Pictorial Map to help you write your own Essay. org 3 | P a g e. Pictorial drawing. The pictorial essay is a semester-long project in which you assemble images to present a topic of your choice. MBA Essay Writing Tips, Essay Topics, Essay Samples, Essay Format, How to write an impressive essay. How to Write a Photo Essay. Official Full-Text Publication: CT enteroclysis: A pictorial essay sample pictorial pictorial essay sample essay on ResearchGate, the professional network for scientists Pictorial Dynamics Analysis In an essay of at least two Be sure you note any disruptions--places where the artist has chosen to do something that defies. The test describes an issue and provides three. Learn about essay outline format and behavior essay for students to copy essay outline structure, review essay outline template, essay outline sample and reserch outline sample Essay topics
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Before kids I was a nice and perky 32B. When I started nursing my first born I became a 32C. Then, after nursing, my size dropped to a wrinkly, deflated size 32A. My son nursed more on one side than the other so on top of that, one boob was larger than the other and my areolas remained large from being a 32C even tho I shrunk in size. After seeing the drastic change, I became self conscious and never, I mean never showed my boobs. I've been with my bf for 5 years and I still have never took my bra off during intimate times. I wanted a boob job then but was never the right moment. Then, I got pregnant (5 years later) and again nursed. This time I nursed for almost 2 years vs 9 months with my first. At largest, my boobs were a Small B. They did not get as large as my first pregnancy/child. After I weaned, recently, my boobs are not even a 32aa. Literally, I wear a 32aa bra and it rides up bc I've lost so much volume and have nothing to hold it down. I'm the epitome of "flat chested". Only now, I decided it's time to fix and restore the fullness so I finally pulled the trigger and put a deposit down and schedule my surgery! I'm sooo excited and terrified (but more excited) that I live and breathe breasts. Literally all day and night I think about boobs and having the perfect boobs and I talk about it. I'm pretty sure everyone is over the conversation but I'm not. So, here I am writing my review and sharing my story. I know many women have shared their stories and I've read the good, the bad, and the ugly. But what's most comforting is the amount of support everyone has and that is why I decided to turn here. I hope to share my support and receive feedback. I'm excited to share my journey. Can't wait for MAY!!
29 Years Old, Petite, 4'11, 90 Pounds, 2 Breastfed Kids - California, CA
Before kids I was a nice and perky 32B. When I...
Less than 2 months left!!
Time couldn't go by any slower when you're waiting for your BA! Lol! Here's a few photos I took the other day and will take and after my BA.
Last time I have GNO pre-BA
Trying on things for a Bach this weekend and everything I try on is so sad. But, I bought 2 padded sticky bras and a padded push up to try and create miracle boobs/cleavage! Here's some items i may wear and will be sure to try them again after my surgery!
3 more weeks!
I'm so excited and it's finally the month I get my ba done! I had a few weeks where I'd be up all day and night staring at wish boobs and forums and now that it's finally closer, I feel another wave coming on. I have my pre op this weekend and so far it hasn't hit me but maybe it will at the pre op! Nonetheless I'm excited and already want to buy new clothes but I gotta wait!
8 days left!!!!
...and time couldn't go by any closer! I've waited 6 years for a BA and now that it's basically week away, it still hasn't hit me that this is happening. However I find that I'm up late looking at boobs and what I should do to prepare. So far all I've done is get new sports bras. Any advice or tips? How much were you in bed bc I'm thinking of getting those portable bed trays for food and magazines. I tore apart my place trying to clean up and throw out stuff that I've been putting off. Anyone else do this? I work in an office and curious if anyone's had a lift/ba and how soon they returned to work. My procedure is next Thursday and I'm returning to work the following Tuesday. I'm rambling and asking so many questions but hurrryyyy time!
2 more days! Experiencing heavy nesting...
I'm only 2 days away and all of a sudden this week I've started nesting, similar to when I was pregnant. I hired housekeepers, got our carpets cleaned, moved around the entire place and re organized, cleaned my closets..has anyone experienced this? Also, I'm having a case of paranoia. I keep saying I hope I'm not pregnant. I don't think I am but the fact that they say don't get pregnant before your BA, I'm scared it could happen and I'd be so sad I wouldn't be able to get my ba. I'm scared I might catch a cold, I'm scared or anything that could cause me to NOT get my ba that I've waited 6 or so years for and I'm getting nausea thinking about it. Ahhhh!!!
Had my surgery today!!!
I did it! I told my dr I Santa small c and I ended up with 275. Minimal pain and he whole staff was amazing! I can't wait to see my results. I'm currently in bed half asleep and still wearing off the drugs but I haven't had any pain meds yet. I'm only gonna take it whe necessary. I'll take pics as soon as I can!!!
Post op day 1
Got my bandages removed today and I'm happy with the results. Dr says I can start massaging in 2 days. I can't wait to see how they dropped. Here's my stats: 4'11 94 pounds 29 275 right and left Mentor round silicone mod +
Post op day 3
Today is 3 days post op and I feel amazing! I must say that I'm in zero pain and have been off pain killers since day 2. My Drs skills were amazing! However, I never understood until now...booby greed. This is in no way my Drs fault, he did exactly as I asked for initially "full B/small C" and then the day of surgery I switched and said "small to mid C" When I came out I felt like I was still the same size and a B at most. I know I have to give it time but I'm being impatient and kinda wishing I went larger (which I hear is typical). So, we will see as the days go on what size I'll end up (fingers crossed). I think I wanted to be conservative but deep deep deep down, maybe I really wanted to be a D (ahhh).
I decided to carefully try on an old top I wore recently after I massaged them and it gave me hope bc they actually looked nice in the photo. I know they're swollen and need to d&f so I'll try it on again in another few months! Take a peak!
One week post op!
Can't believe it!
Healing has been a breeze so far. Still haven't used any pain meds although yesterday I experienced some slight nerve pain around my incision area. I attribute this to the healing process, dealt with it and it disappeared. Here's a progress pic!
Healing has been a breeze so far. Still haven't used any pain meds although yesterday I experienced some slight nerve pain around my incision area. I attribute this to the healing process, dealt with it and it disappeared. Here's a progress pic!
10 days post op
Have my 10 day follow up tomorrow and I'm feeling great. I realized that the surgery threw off my menstrual cycle because im usually spot on but I'm a few days late. Has anyone experienced this?
2 weeks and 3 days post op!
My left pocket doesn't as tight as the right. If you take a look, the crease isn't as defined. However it appears my right breast hasn't dropped as much as my left. I know I'm still healing and overall, time will tell. As far as my incisions, they seem to be healing just fine and I'm looking forward to applying creams soon for the scarring!
5 weeks and 4 days!
They're so much softer and getting softer everyday. They do seem smaller and I still wish they were bigger but I can see they're dropping and changing shape.
I'm able to lift my arms and lift now and exercise lightly.
As for my incisions, I don't put anything on them and they've healed great so far and my scars aren't raised, they are blending in. Once my last few stitches completely heal I'll put aquaphor or coconut oil but definitely minimal products.
I'm able to lift my arms and lift now and exercise lightly.
As for my incisions, I don't put anything on them and they've healed great so far and my scars aren't raised, they are blending in. Once my last few stitches completely heal I'll put aquaphor or coconut oil but definitely minimal products.
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A national gathering of Intentional Eucharistic Communities is scheduled to open in in Chevy Chase, MD., tomorrow. I recently talked with William D'Antonio -- a professor of the sociology of religion and well-regarded researcher -- about the gathering. D'Antonio is the principal organizer of the gather. My interview with D'Antonio is an NCR podcast: Intentional communities find a way
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My wife and I were looking for a 3-night stay in Santaigo to celebrate our wedding anniversary. We flew in from Atlanta, GA, Thursday night, arriving Friday morning. I am actually writing this review, while sitting at the dinning table at Hotel Orly. The hotel staff was very friendly and accomodating. We elected to stay off-site of the main hotel because it offered a small apartment size stay vs. the single rooms in the main building, that is located about a half of a block down the street. For stays longer than one night, this is the way to go.
The only reason I'm not giving a 5-star is due to the big-screen TV in the living room, was not working because the TV's remote couldn't be located. The apartment is spacious enough for two couples or a family. With microwave, washer, refrig. Comes with cookware, dishes and fee bottled water, coffee and tea. Overall we are very pleased with everything including the price!
- Reservation Options:
- TripAdvisor is proud to partner with Booking.com, Orbitz, Expedia, Hotels.com, TripOnline SA, Priceline, Travelocity, Hotwire, Cancelon, getaroom.com and Evoline ltd so you can book your Hotel Orly reservations with confidence. We help millions of travelers each month to find the perfect hotel for both vacation and business trips, always with the best discounts and special offers.
- Also Known As:
- Orly Hotel Santiago


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HOULTON – James A. Logue Sr., 86, died May 24, 2004, at a local hospital. He was born Sept. 14, 1917, at New Limerick, the son of Charles and Effie (Henderson) Logue. Jim served as a Sea-bee in the U.S. Navy during World War II. He was employed by Harry Bass Farms for many years. He is survived by one son, James Logue Jr. of Houlton; four daughters, Bonnie Reardon of Rodchester, N.H., Sandra Logue of Monticello, Shireen Belyea and Debra Logue, both of Houlton; 20 grandchildren; 39 great-grandchildren; seven great-great-grandchildren; a special friend, Lillian Stairs of Houlton. He was predeceased by his wife, Kathryn Logue, in 1995. At Jim's request there will be no services. Interment was in the Evergreen Cemetery. A service of Dunn Funeral Home.
Posted Sept. 25, 2008, at 11:14 p.m.


text: This monster article is a stub. You can help the Diablo Wiki to become more awesome by expanding it. Cloud Stalkers appear in Act III. This is stronger and colored blue version of a Foul Crow. On Hell, Cloud Stalkers are immune to cold. There're no Cloud Stalker Nests in the game. Blood Hawks v t e Foul Crow Blood Hawk Black Raptor Cloud Stalker Retrieved from "http://diablo.wikia.com/wiki/Cloud_Stalker?oldid=77150" Ad blocker interference detected! Wikia is a free-to-use site that makes money from advertising. We have a modified experience for viewers using ad blockers Wikia is not accessible if you've made further modifications. Remove the custom ad blocker rule(s) and the page will load as expected. Categories: Monster article stubs Monsters Diablo II Bestiary Blood Hawk Add category Cancel Save


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South Korea's top envoy to North Korea heads to US
South Korea's top envoy on North Korea left on Tuesday for a meeting in Washington with his US and Japanese counterparts, two days after Pyongyang proposed direct, high-level talks with the United States.
After Washington, Cho Tae-yong will travel on to Beijing - where he is likely to overlap with a visit by North Korea's chief nuclear negotiator, Kim Kye-gwan, to the Chinese capital.
Speaking to the South's Yonhap news agency before his departure, Cho said it was important to retain a clear perspective on the North's recent moves towards dialogue after months of military tensions.
"At this point, it is important for relevant parties to rebuild trust so that progress can be made toward the goal of denuclearising North Korea, rather than talks for talks' sake," Cho said.
The North's offer of direct denuclearisation talks with Washington - bypassing any initial dialogue with Seoul - was seen by many analysts as a bid to drive a wedge between the United States and its South Korean ally.
The US responded by insisting that Pyongyang first demonstrate its willingness to abandon its nuclear weapons programme.
North and South Korea had been scheduled to hold their first high-level talks for six years last week, but they were cancelled after a row over protocol.
In Washington, Cho will meet with the US and Japanese special envoys on North Korea, Glyn Davies and Shinsuke Sugiyama, but said he had "no plan" to meet Kim Kye-gwan in Beijing.
South Korean President Park Geun-hye is due to make her first visit to China later this month for a summit with President Xi Jinping.
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Cut The Rope 2
Cut The Rope 2 is a free Physics Games. Here comes the fun gam Cut the Rope 2, which is returned with many more levels, currently 300 to be exact and more coming soon. Your mission is to cut the candies hanging from ropes in each level and try to collect all the stars to unlock new levels. You can use the money you get to buy hints, power-ups, and customize your cute monster the shopping cart.Have fun! If you like Cut The Rope 2, you can put it to your favorites. So it can be convenient for you to play later!
You can play the game on smartphone and tablet (iPhone, iPad, Samsung, Android devices and Windows Phone)
We hope you had fun with this great Puzzle Games. Find the best Physics Games among our games below.
Published: 12-04-2015
672 Users Played
How to play Cut The Rope 2 game?
Detailed instructions can be found in the game or in description or use Mouse/keyboard to interactive.
Why Can't We Play This Game?
Cut The Rope 2 is an online Physics Games which you can play for free here at ChipGames.net - It has tag(s) Physics Games, HTML 5 Games, Kids Games, Mobile and Tablet Games, Puzzle Games. It has been played 672 times. Cut The Rope 2 is one of the many Puzzle Games that we feature. If you want to play more games, check out Cut The Rope game or Cut the Rope: Time Travel game or Cut The Rope Experiments game or Short Cut game or Cute Cookie Cut game or Ben 10 Cut Fruit game or Rope Ninja game or Fly With Rope 2 game. To play even more games, head over to the Physics Games page, the HTML 5 Games page, the Kids Games page, the Mobile and Tablet Games page, the Puzzle Games page or just check out the all time best games page.


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The interaction of Kinesin-1 with its adaptor protein JIP1 can be regulated via proteins binding to the JIP1-PTB domain
Cytoplasmic and organelle biology
The regulatory mechanisms of motor protein-dependent intracellular transport are still not fully understood. The kinesin-1-binding protein, JIP1, can function as an adaptor protein that links kinesin-1 and other JIP1-binding "cargo" proteins. However, it is unknown whether these "cargo" proteins influence the JIP1–kinesin-1 binding.
We show here that JIP1–kinesin-1 binding in Neuro2a cells was dependent on conserved amino acid residues in the JIP1-phosphotyrosine binding (PTB) domain, including F687. In addition, mutation of F687 severely affected the neurite tip localization of JIP1. Proteomic analysis revealed another kinesin-1 binding protein, JIP3, as a major JIP1 binding protein. The association between JIP1 and JIP3 was dependent on the F687 residue in JIP1, and this association induced the formation of a stable ternary complex with kinesin-1. On the other hand, the binding of JIP1 and JIP3 was independent of kinesin-1 binding. We also show that other PTB binding proteins can interrupt the formation of the ternary complex.
The formation of the JIP1–kinesin-1 complex depends on the protein binding-status of the JIP1 PTB domain. This may imply a regulatory mechanism of kinesin-1-dependent intracellular transport.
Kinesin-1, also known as conventional kinesin, is a member of the Kinesin super family proteins (KIFs). KIFs move along microtubules (MTs) by ATP hydrolysis and are essential for the transport of a variety of cargos such as protein complexes and membranous organelles
JNK interacting protein (JIP) 1 binds to the KLC through its C-terminal kinesin-binding site
In this study, we tested the significance of JIP1 binding proteins for the formation of the JIP1–kinesin-1 complex in mammalian cells. We demonstrated that conserved amino acid residues in the PTB domain, including F687, but not the JBD of JIP1 enhance the formation of a stable complex with kinesin-1, while the C-terminal residues show an absolute requirement for this interaction. We then identified another kinesin-1 binding protein, JIP3, responsible for the F687-dependent enhancement of the formation of the JIP1–kinesin-1 complex. We further analyzed the molecular basis of the enhancement of JIP1–kinesin-1 complex formation. The results not only suggest a regulatory role of JIP3 in the formation of the JIP1–kinesin-1 complex, but also suggest a possible regulatory mechanism mediated by JIP1-binding proteins that bind to the JIP1-PTB domain.
Formation of the JIP1–kinesin-1 complex in Neuro2a cells is independent of the JIP1-JBD and cellular JNK activity
To examine the requirement of JIP1 binding proteins for the association between JIP1 and kinesin-1, we made a series of deletions or amino acid substitutions in the JBD and PTB domains of JIP1 (Figure
Figure 1
Formation of the JIP1–kinesin-1 complex in Neuro2a cells is dependent on the JIP1-PTB domain but not JIP1-JBD
Formation of the JIP1–kinesin-1 complex in Neuro2a cells is dependent on the JIP1-PTB domain but not JIP1-JBD. (A). Schematic illustration of the JIP1 constructs used in this study. The N-terminal 276 residues were deleted in dJBD. The C-terminal 4 residues were deleted in dCT. V581, F642 and F687 were substituted by A or V in V581A, F642V and F687V. JBD: JNK binding domain; SH3: src homology 3 domain; PTB: phosphotyrosine binding domain; Y705: required tyrosine for kinesin-1 binding. These constructs are GFP- or TAP-tagged at their N-termini. (B). Lysates prepared from Neuro2a cells expressing GFP-JIP1 constructs or GFP alone were immunoprecipitated with anti-GFP antibody and analyzed by western blotting (WB) with the indicated antibodies.
Additional file 1: Figure S1
JNK activity does not affect JIP1 binding to kinesin-1. (A). Co-precipitation of GFP-JIP1 mutants and endogenous JNK in Neuro2a cells. The same samples as shown in Fig. 2A were analyzed by WB with the indicated antibodies. (B). Neuro2a cells transiently expressing TAP-JIP1 were treated with SP600125 (20 μM) or DMSO (vehicle) for 2 hours. Lysates were precipitated with SA.
Click here for file
A possible role for JNK in the interaction between JIP1 and kinesin-1 has been proposed. In
The PTB domain of JIP1 is required for complex formation with kinesin-1
We next examined the requirement of the PTB domain for the association between JIP1 and kinesin-1. Because deletion of the JIP1 PTB domain resulted in insoluble aggregate formation in Neuro2a cells, we used amino acid substitution to generate PTB domain mutants that lacked protein-binding activity (Figure
Additional file 2: Figure S2
Sequence alignments of the JIP1 PTB domain. (A) JIP1 PTB domain aligned with Shc- and Dab1-PTB domains. All sequences are from mouse. The gray shading indicates the amino acids corresponding to JIP1 V581, F642 and F687. (B). Alignment of JIP1 PTB domains between species. The gray shading indicates the amino acids corresponding mouse JIP1 V581, F642 and F687.
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Identification of JIP3 as a principal binding protein of JIP1
The V581, F642 and F687 mutations in the PTB domain drastically reduced the binding of JIP1 to kinesin-1. Because the JIP1-PTB domain cannot directly interact with kinesin-1
Figure 2
Identification of JIP3 as a major PTB domain-dependent binding protein of JIP1
Identification of JIP3 as a major PTB domain-dependent binding protein of JIP1. (A). Neuro2a cells transiently transfected with either TAP-JIP1 or control vector were used for pull-down experiments with streptavidin sepharose (SA). The JIP1 associated polypeptides were visualized by silver staining. (B). Co-precipitation of GFP-JIP1 constructs and endogenous JIP3 in Neuro2a cells. The same samples as shown in Fig. 1B were analyzed by WB with the indicated antibodies. (C). Lysates of HEK293T cells expressing TAP-JIP1, V5-tagged kinesin light chain (V5-KLC), V5-KHC, GFP-JIP3, DLK, APP, or GFP alone were precipitated with SA and analyzed by WB with the indicated antibodies.
Additional file 3: Figure S3
Mouse JIP3 peptides identified by Mascot search (Matrix Science). Amino acid sequence of mouse JIP3. Capital letters shaded gray indicate the peptides identified by Mascot search.
Click here for file
To test this possibility, we performed co-precipitation assays using lysates from HEK293T cells, which did not contain detectable levels of JIP3, JIP1, and kinesin-1 proteins. Cells were transfected with expression vectors for TAP-tagged JIP1, V5-tagged KLC, and V5-tagged KHC, together with an expression vector for GFP-JIP3 or GFP. As shown in Figure
Although we were able to identify JIP3 as a major JIP1 PTB domain binding protein in differentiated Neuro2a cells, several proteins other than JIP3 have been reported as JIP1 PTB domain binding proteins in other cell systems
The association of JIP1 with JIP3 and kinesin-1 was evaluated
JIP3 enhances the association between JIP1 and kinesin-1 in Neuro2a cells
To test whether JIP3–JIP1 binding is essential for the association between JIP1 and kinesin-1, we investigated the effect of JIP3 knockdown. Neuro2a cells stably expressing RFP-tagged JIP1 (RFP-JIP1-Neuro2a cells) were transiently transfected with expression vectors for JIP3 shRNA (shJIP3) or non-silencing shRNA (NS). Immunoprecipitation of RFP-JIP1 followed by western blotting using anti-KHC antibody revealed that the binding of RFP-JIP1 and kinesin-1 was severely reduced by JIP3 knockdown (Figure
Figure 3
JIP1 requires JIP3 for binding to kinesin-1 in Neuro2a cells
JIP1 requires JIP3 for binding to kinesin-1 in Neuro2a cells. (A). Lysates prepared from RFP-JIP1-Neuro2a cells expressing shRNA targeting JIP3 (shJIP3) or non-silencing control (NS) shRNA were immunoprecipitated with anti-RFP antibody and analyzed by WB with the indicated antibodies.
Additional file 4: Figure S4
The effect of JIP3 knockdown was offset by JIP3-WT but not JIP3-LZ4A. (A). Lysates prepared from RFP-JIP1-Neuro2a cells expressing shRNA targeting JIP3 (shJIP3), non-silencing control (NS) shRNA, TAP-JIP3-WT, or TAP-JIP3-LZ4A, in the combination as indicated were immunoprecipitated with anti-RFP antibody and analyzed by WB with the indicated antibodies. Input, cell lysate used for the immunoprecipitation assay. IP:RFP, immunoprecipitated proteins. (B). Quantification of kinesin-1 binding by RFP-JIP1 in (A). Kinesin-1 binding was normalized to the amount of precipitated RFP-JIP1 and input of kinesin-1. Results of two independent experiments are shown. (C). Differentiated RFP-JIP1-Neuro2a cells were transfected with shRNA vectors (NS or shJIP3) containing a GFP expression cassette. Arrowheads indicate the neurite tips of transfected cells. Scale bar = 20 μm. (D). Quantification of the relative fluorescence of RFP-JIP1 in the neurite tip. *: p < 0.03. Error bars indicate ± SEM. n = 50 for each construct.
Click here for file
Molecular basis for JIP1–JIP3–kinesin-1 ternary complex formation
As shown in Figure
We confirmed that roughly equal amounts of V5-KHC and V5-KLC were present in the JIP1 pull-down fraction (see Figure
Figure 4
Molecular basis for JIP1–JIP3–kinesin-1 ternary complex formation
Molecular basis for JIP1–JIP3–kinesin-1 ternary complex formation. (A). Lysates prepared from HEK293T cells expressing TAP-JIP1, V5-KLC, V5-KHC and GFP-JIP3, or GFP alone, were precipitated with SA and analyzed by WB with the indicated antibodies.
We then sought to clarify why the JIP1–JIP3 complex showed such higher kinesin-1-binding activity. Because both JIP1 and JIP3 can bind to kinesin-1
Finally, we examined whether the JIP1–JIP3 complex is stabilized by kinesin-1. As shown in Figure
DLK affects the formation of the JIP1–JIP3–kinesin-1 ternary complex
The results presented above suggest the presence of a JIP1–JIP3–kinesin-1 ternary complex. We then tested whether the formation of the ternary complex was regulated by an additional factor, which may serve as a regulator of the JIP1–JIP3–kinesin-1 complex formation. Because the formation of the JIP1–JIP3–kinesin-1 ternary complex was highly dependent on PTB domain-dependent binding of JIP1 and JIP3, we hypothesized that other PTB domain-dependent JIP1 binding proteins would interrupt the formation of the ternary complex. If the PTB binding protein has no kinesin-1 binding activity, the binding of kinesin-1 to this protein complex will be restricted. One of the candidates for such a protein is DLK, which is reported to stably interact with JIP1 via the PTB domain, particularly when its kinase activity is suppressed
Figure 5
DLK affects the formation of the JIP1–JIP3–kinesin-1 ternary complex
DLK affects the formation of the JIP1–JIP3–kinesin-1 ternary complex. (A). Lysates of Neuro2a cells expressing TAP-JIP1 and T7-DLK-WT, T7-DLK-KR or pUC8 were precipitated with SA and analyzed by WB with the indicated antibodies.
In this study, we investigated the contribution of JIP1 binding proteins to the interaction between JIP1 and kinesin-1. Although the binding of JNK to the JBD of JIP1 showed no significant effect on the kinesin-1 binding, the binding of JIP3 to the PTB domain of JIP1 enhanced it significantly. DLK, another JIP1-PTB domain binding protein, did not show such an effect. However, the over-expression of kinase-inactive DLK perturbed the formation of the JIP1–JIP3–kinesin-1 ternary complex, suggesting that DLK might compete with JIP3 for the JIP1-PTB domain. Taken together with the reported function of JIP1 as a cargo adaptor
JIP1 and JIP3 were originally identified as JNK binding proteins that function as a scaffold for the JNK activating protein kinase cascade in mammalian cells
In contrast to the dispensability of the JIP1 JBD, the PTB domain is essential for kinesin-1 binding by JIP1 in differentiated Neuro2a cells. We identified JIP3 as a major PTB-dependent JIP1 binding protein that was largely essential for the neurite tip localization and kinesin-1-binding of JIP1. JIP1 or JIP3 bound to kinesin-1 via their kinesin-1-binding motifs, without forming a JIP1–JIP3 complex. However, this "basal" kinesin-1 binding activity was significantly enhanced by JIP1–JIP3 complex formation, which was totally dependent on the JIP1-PTB domain. A previous study showed that distinct regions of the TPR domains in KLC are responsible for JIP1 and JIP3 binding
Because the kinesin-1 binding activity of JIP3 is crucial for the formation of the ternary complex, other proteins that compete with JIP3 for binding to the JIP1-PTB domain and lack the capacity to bind kinesin-1 may negatively regulate the potency of JIP1 for kinesin-1 binding. Using DLK, we demonstrated a potential regulatory role for JIP1-PTB domain binding proteins in the formation of the JIP1–JIP3–kinesin-1 ternary complex. In addition, kinesin-1 or JIP3-binding proteins may also affect the formation of the JIP1–JIP3–kinesin-1 complex. The inhibition of JIP1–kinesin-1 binding by Ca2+−dependent binding of S100A6 to KLC, and the inhibition of JIP3–kinesin-1-binding by GTP-dependent binding of ARF6 to JIP3, have been reported
In this study, we have shown that JIP3 is a major binding protein of JIP1 in Neuro2a cells, and that JIP3 has the highest JIP1-binding capacity among various known JIP1-PTB domain binding proteins. Because JIP1 and JIP3 are highly expressed in neurons
We investigated the contribution of JIP1 binding proteins to the interaction between JIP1 and kinesin-1. The binding of JIP3 to the PTB domain of JIP1 enhanced the JIP1–kinesin-1 interaction significantly, while the binding of JNK to the JBD of JIP1 showed no significant effect. DLK, another JIP1-PTB domain binding protein, did not show any effect on JIP1–kinesin-1 binding by itself. However, the over-expression of kinase-inactive DLK perturbed the formation of the JIP1–JIP3–kinesin-1 ternary complex, suggesting that DLK might compete with JIP3 for the JIP1-PTB domain. Our results suggest that the proteins that bind to the JIP1-PTB domain function as regulators of JIP1–kinesin-1 complex formation.
HEK293T human embryonic kidney cells, Neuro2a mouse neuroblastoma cells, and RFP-JIP1 stably transfected Neuro2a cells (RFP-JIP1-Neuro2a) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS). The cell lines were cultured under 5% CO2 at 37°C. For differentiation, Neuro2a and RFP-JIP1-Neuro2a cells were incubated with medium containing 5% FBS and 20 μM all-trans-retinoic acid (Wako) for 24–48 h.
For transfection, 3 × 105 HEK293T or 6 × 105 Neuro2a cells were plated in 6-cm dishes. After 48 h, the cells were transfected with 5–10 μg of plasmid using Lipofectamine LTX (Invitrogen). After 24 h, the cells were used for co-precipitation assays. For knock-down of JIP3, 1 × 106 RFP-JIP1-Neuro2a cells were transfected with 3–5 μg of plasmids using either Nucleofector II (Amaxa) or Neon (Invitrogen) techniques, and plated in 6-cm dishes. After 48 h, the cells were used for co-precipitation assays. For immunofluorescence, Neuro2a and RFP-JIP1-Neuro2a cells were plated in 24-well plates for 24 h. The culture medium was changed to differentiation medium and the cells were incubated for a further 24 h. The cells were transfected with 0.5 μg of plasmids using Lipofectamine LTX. After 24–30 h, the cells were processed for immunofluorescence staining.
Immunofluorescence and neurite tip measurements
Neuro2a and RFP-JIP1-Neuro2a cells were fixed with 3% paraformaldehyde in PBS for 15 min and washed three times with PBS. Following permeabilization with 0.1% Triton X-100 in PBS for 10 min, the cells were washed three times with PBS and quenched with 0.1 M glycine in PBS for 30 min. The cells were then washed a further three times with PBS and blocked with 10% calf serum in TBST for 1 h. All of these steps were performed at room temperature. The cells were then incubated in 0.1% BSA in TBST containing primary antibodies for 16–48 h at 4°C, washed three times with TBST, and labeled with Alexa 488-, 546-, or 647-conjugated anti-mouse or anti-rabbit antibodies. Quantification of neurite tip fluorescence intensity was performed using Multi Gauge software (Fujifilm). The neurite tip and its shaft were hand-selected with the rectangle tool, and the average pixel fluorescence intensity was measured. The average intensity ratio between the tip and shaft of individual neurites was employed as the localization value.
Co-precipitation assays
All of the following steps were performed at 4°C. The cells were washed twice with ice-cold PBS and lysed in 0.5 ml of lysis buffer (150 mM NaCl, 20 mM Hepes, pH 7.5, 1 mM EGTA, 50 mM NaF, 25 mM beta-glycerophosphate, 1 mM Na3VO4, pH 10, 5 μg/ml aprotinin, 10 μg/ml leupeptin, 0.5% Triton X-100, 1% protease inhibitor cocktail (Sigma)) for 10 min on ice. The lysates were then centrifuged at 15,000 × g for 20 min, and the resulting supernatants were used for co-precipitations. The supernatants were incubated with either 1 μg of antibody or Streptavidin Sepharose (GE Healthcare) for 90 min, following which the immunocomplexes were recovered with Protein A Sepharose (BD Biosciences Pharmingen). The Streptavidin Sepharose and Protein A Sepharose pellets were then washed three times with lysis buffer and boiled with SDS sample buffer.
Two whole brains from ICR E17 mouse embryos were Dounce-homogenized 10 times in 1 ml of lysis buffer and incubated on ice for 10 min. The homogenate was then centrifuged at 15,000 × g for 20 min, and the resulting supernatants were used for co-precipitations. The supernatants were incubated with either 8 μg of anti-JIP1 antibody or normal rabbit IgG for 4 hours, following which the immunocomplexes were recovered with Protein A Sepharose. The Protein A Sepharose pellets were then washed three times with lysis buffer and boiled with SDS sample buffer.
JIP1, JIP3, KLC, KHC, APP, MEKK1 and DLK expression vectors
The mouse JIP-1b cDNA (encoding a polypeptide of 707 aa) was cloned into the pEGFP-C vector (Clontech) and the pNTAP vector (Stratagene) (GFP-JIP1-WT and TAP-JIP1-WT, respectively). GFP-JIP1-WT was digested with BglII and self-ligated to generate the GFP-JIP1-dJBD vector. The GFP-JIP1-WT and TAP-JIP1-WT vectors were digested with EcoRV and ligated with a stop codon linker to generate the GFP-JIP1-dCT and TAP-JIP1-dCT vectors. As the first step in the generation of the GFP-JIP1-V581A vector, the JIP1-WT vector was digested with StuI and EcoRV, and the fragment was sub-cloned into pBlueScript KS (JIP1-PTB-pBS). Silent mutations were introduced by site-directed mutagenesis PCR to generate BsrGI and NruI sites at 571 and 590, respectively. The synthetic complementary oligonucleotides containing the V581A mutation were cloned into the BsrGI-NruI site. Then, the resultant vector was digested with BstEII and EcoRV, and the fragment was cloned into GFP-JIP1-WT. GFP-JIP1-F642V and F687V were produced by site-directed mutagenesis PCR. GFP-JIP1-V581A, -F642V and -F687V mutants were digested with BstEII and EcoRV, and the fragments were cloned into the TAP-JIP1-WT vector. Mouse JIP3-c (1337aa) was cloned into pEGFP and pNTAP vectors (GFP-JIP3-WT and TAP-JIP3-WT, respectively). GFP-JIP3-LZ4A was produced by site-directed mutagenesis PCR. GFP-JIP3-LZ4A was digested with HindIII and BglII, and the fragment was cloned into the TAP-JIP3-WT vector (TAP-JIP3-LZ4A). Mouse KLC-1d (608 aa) and KHC (KIF5C) (957 aa) were cloned into the pCMV-V5 vector (V5-KLC and V5-KHC, respectively). The T7-MEKK1, T7-DLK-WT and T7-DLK-KR vectors were described previously
Short hairpin RNA expression vectors
A shRNA (short hairpin RNA) plasmid targeting mouse JIP3 was constructed using DNA oligos designed with a 19-mer sense sequence, a nine-nucleotide loop, and a 19-mer antisense sequence (sense: 5′-gatccccGGTCTTG ACCCAACATGAAttcaagagaTTCATGTTGGGTCAAGA CCttttta-3′, and antisense: 5′-tcgataaaaaGGTCTTGACC CAACATGAAtctcttgaaTTCATGTTGGGTCAAGACCG GG-3′). Annealed oligos were cloned into the BglII and SalI sites of the pSuper.gfp/neo vector (OligoEngine).
The following antibodies were used: anti-JIP1 mAb (TDL), anti-JIP1 pAb (Zymed), anti-KHC mAb (Chemicon), anti-JIP3 pAb (Santa Cruz), anti-DLK pAb (described previously
Western blotting
Proteins were separated by SDS-PAGE and analyzed by western blotting according to standard protocols. Horseradish peroxidase-conjugated secondary antibodies were used to detect bound primary antibodies. Signal intensities were quantified from the original data recorded by a LAS-4000 Multi Luminescent Image Analyzer and Multi Gauge software (Fujifilm).
Mass spectrometry analysis
Neuro2a cells (~5 × 107 cells) were transfected with the TAP-JIP1 expression vector. One day after transfection, retinoic acid (all-trans, Sigma) was added to the culture medium to a final concentration of 20 μM. Cells were further cultured for 2 days before lysing in 6 ml of the lysis buffer used for the co-precipitation assays (see above). The cell lysate was clarified by centrifugation at 100,000 × g for 30 min, and TAP-JIP1 binding protein was collected as described for the co-precipitation assays. The TAP-JIP1 binding proteins were separated by SDS-PAGE and detected by silver staining for imaging or by SYPRO Ruby (Molecular Probes) for mass spectrometry. The band of interest was cut out and digested with trypsin. The eluted peptides were analyzed on a 4800 MALDI TOF/TOF system (Applied Biosystems).
JIP: c-Jun N-terminal kinase (JNK) interacting protein;PTB: Phosphotyrosine binding;KHC: Kinesin heavy chain;KLC: Kinesin light chain;APP: Amyloid precursor protein;DLK: Dual leucine zipper bearing kinase;WT: Wild type
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
TS and SH designed the experiments. TS and KO performed and analyzed the immunoprecipitation assays. TS performed and analyzed the immunofluorescence assays. HH and YY performed the mass spectrometry analysis. TS, KO, YB, JS and SH constructed the expression vectors. TS, SH and SO wrote the manuscript. All authors read and approved the final manuscript.
We thank Dr. T.C. Saido for kindly providing the human APP cDNA, and Dr. I. Nishimoto for the mouse JIP1 cDNA. This work was supported by JSPS KAKENHI Grant Number 21570184.
Intracellular transport and kinesin superfamily proteins, KIFs: structure, function, and dynamics
Kinesin superfamily motor proteins and intracellular transport
Cargo of kinesin identified as JIP scaffolding proteins and associated signaling molecules
APLIP1, a kinesin binding JIP-1/JNK scaffold protein, influences the axonal transport of both vesicles and mitochondria in Drosophila
Control of a kinesin-cargo linkage mechanism by JNK pathway kinases
A cytoplasmic inhibitor of the JNK signal transduction pathway
Jun NH2-terminal kinase (JNK) interacting protein 1 (JIP1) binds the cytoplasmic domain of the Alzheimer's beta-amyloid precursor protein (APP)
Interaction of c-Jun amino-terminal kinase interacting protein-1 with p190 rhoGEF and its localization in differentiated neurons
The reelin receptor ApoER2 recruits JNK-interacting proteins-1 and −2
Mixed lineage kinase LZK forms a functional signaling complex with JIP-1, a scaffold protein of the c-Jun NH(2)-terminal kinase pathway
Co-operative versus independent transport of different cargoes by Kinesin-1
Structural and evolutionary division of phosphotyrosine binding (PTB) domains
Differential roles of JIP scaffold proteins in the modulation of amyloid precursor protein metabolism
Amyloid beta protein precursor (AbetaPP), but not AbetaPP-like protein 2, is bridged to the kinesin light chain by the scaffold protein JNK-interacting protein 1
Activation of the JNK pathway by distantly related protein kinases. MEKK and MUK
JLP associates with kinesin light chain 1 through a novel leucine zipper-like domain
Recruitment of JNK to JIP1 and JNK-dependent JIP1 phosphorylation regulates JNK module dynamics and activation
A mammalian scaffold complex that selectively mediates MAP kinase activation
JSAP1, a novel jun N-terminal protein kinase (JNK)-binding protein that functions as a Scaffold factor in the JNK signaling pathway
Unloading kinesin transported cargoes from the tubulin track via the inflammatory c-Jun N-terminal kinase pathway
Scaffold protein JSAP1 is transported to growth cones of neurites independent of JNK signaling pathways in PC12h cells
Sunday Driver/JIP3 binds kinesin heavy chain directly and enhances its motility
Two binding partners cooperate to activate the molecular motor Kinesin-1
Interactions of S100A2 and S100A6 with the tetratricopeptide repeat proteins, Hsp90/Hsp70-organizing protein and kinesin light chain
ARF6 Interacts with JIP4 to control a motor switch mechanism regulating endosome traffic in cytokinesis
Expression of JNK cascade scaffold protein JSAP1 in the mouse nervous system
Spatial, temporal and subcellular localization of islet-brain 1 (IB1), a homologue of JIP-1, in mouse brain
Kinesin-dependent axonal transport is mediated by the sunday driver (SYD) protein
UNC-16, a JNK-signaling scaffold protein, regulates vesicle transport in C. elegans
The JIP1 scaffold protein regulates axonal development in cortical neurons
The scaffold protein JIP3 functions as a downstream effector of the small GTPase ARF6 to regulate neurite morphogenesis of cortical neurons
MAPK-upstream protein kinase (MUK) regulates the radial migration of immature neurons in telencephalon of mouse embryo
The c-Jun N-terminal kinase activator dual leucine zipper kinase regulates axon growth and neuronal migration in the developing cerebral cortex


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7. Fresh fruit
Fresh fruit has a very short shelf life and shockingly we waste 1.4 million whole bananas a year!
How to avoid waste Just because bananas are ripe, it doesn't mean they should be heading for the bin. Mushy and ripe is exactly how you need bananas for baking as they produce a much moister sponge - try our classic banana bread recipe. You can also freeze bananas and defrost in the microwave or blend down for a lower-fat ice cream alternative.
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We make cakes with ours or fry them (with or without batter) and eat them hot with golden syrup.....drool
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- Slave \Slave\, v. i. [imp. & p. p. {Slaved}; p. pr. & vb. n. {Slaving}.] To drudge; to toil; to labor as a slave. [1913 Webster]
The Collaborative International Dictionary of English. 2000.
The Collaborative International Dictionary of English. 2000.
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