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A-Kinase Anchoring Protein Targeting of Protein Kinase A and Regulation of HERG Channels
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Adrenergic stimulation of the heart initiates a signaling cascade in cardiac myocytes that increases the concentration of cAMP. Although cAMP elevation may occur over a large area of a target-organ cell, its effects are often more restricted due to local concentration of its main effector, protein kinase A (PKA), through A-kinase anchoring proteins (AKAPs). The HERG potassium channel, which produces the cardiac rapidly activating delayed rectifying K+ current (I Kr), is a target for cAMP/PKA regulation. PKA regulation of the current may play a role in the pathogenesis of hereditary and acquired abnormalities of the channel leading to cardiac arrhythmia. We examined the possible role for AKAP-mediated regulation of HERG channels. Here, we report that the PKA-RII-specific AKAP inhibitory peptide AKAP-IS perturbs the distribution of PKA-RII and diminishes the PKA-dependent phosphorylation of HERG protein. The functional consequence of AKAP-IS is a reversal of cAMP-dependent regulation of HERG channel activity. In further support of AKAP-mediated targeting of kinase to HERG, PKA activity was coprecipitated from HERG expressed in HEK cells. Velocity gradient centrifugation of solubilized porcine cardiac membrane proteins showed that several PKA-RI and PKA-RII binding proteins cosediment with ERG channels. A physical association of HERG with several specific AKAPs with known cardiac expression, however, was not demonstrable in heterologous cotransfection studies. These results suggest that one or more AKAP(s) targets PKA to HERG channels and may contribute to the acute regulation of I Kr by cAMP.
KeywordsPKA AKAP HERG Potassium channel Phosphorylation Protein interaction
Dynamic control of action potentials in cardiac tissue underlies the normal response to varying physiological demands placed on the heart. In large part, this is governed by changes in ion channel activity in response to the autonomic nervous system cholinergic and adrenergic stimuli commensurate to hemodynamic challenges. Dysregulation of this balance plays a central role in pathological conditions such as heart failure. Spatial and substrate specificity of β-adrenergic signaling is maintained by localization of protein kinase A (PKA) and phosphodiesterases to subcellular microdomains. Localization of these proteins may be achieved by scaffolding adapter proteins termed A-kinase anchoring proteins (AKAPs) (Colledge and Scott 1999; Rubin 1994; Ruehr et al. 2004). The AKAPs are a group of proteins that lack primary structure sequence homology but share function: to localize PKA to subcellular structures, substrates, and oftentimes with other members of the signaling pathway (Smith et al. 2006). The regulatory subunits (RI and RII) of PKA bind and hold the catalytic subunits (C) in an inactive state (R2C2 holoenzyme) until they bind cAMP, whereupon active catalytic portions dissociate toward target proteins (Taylor et al. 1990). The R-subunits bind to their respective RI or RII-specific AKAPs, providing local targeting of the catalytic subunit prior to its activation. The N termini of R-subunits form helix-turn-helix structures that create a binding groove for AKAPs, with specificity determined, in part, by the shallow (RII) or deep (RI) nature of the groove (Banky et al. 2003; Gold et al. 2006).
The human ether-a-go-go related gene (HERG) encodes the voltage-dependent potassium channel HERG and produces the repolarizing current I Kr (Sanguinetti et al. 1995). I Kr is important in controlling the orderly repolarization at the end of each cardiac action potential, particularly near the threshold potential of early afterdepolarizations that can trigger tachyarrhythmias. The importance of HERG/I Kr in adrenergic signaling is highlighted by the fact that disease-causing mutations of HERG often lead to a syndrome in which arrhythmias are triggered by sudden emotional or auditory stress (Schwartz et al. 2001; Wilde et al. 1999). HERG/I Kr responds to acute adrenergic signaling in a complex fashion that includes direct cAMP binding to the channel, PKA-dependent phosphorylation of HERG and phosphorylation-dependent binding to the adaptor protein 14-3-3 (Choe et al. 2006; Kagan et al. 2002; Thomas et al. 2003).
Here, we report the first evidence that acute cAMP/PKA-dependent regulation of HERG is targeted by cellular AKAP(s) to determine the degree of channel phosphorylation and biophysical response to acute cAMP stimulation. Such targeted PKA signaling to HERG may represent potential targets for future therapies in both hereditary and acquired dysregulation of the channel.
Materials and Methods
Cell Culture, Transfection and Expression Constructs
The HEK-293 cell line (from American Type Culture Collection, Manassas, VA) stably expressing C-terminal c-myc epitope-tagged HERG cDNA in pCI-Neo vector (Promega, Madison, WI) was cultured in RPMI-1640 (Mediatech, Manassas, VA) supplemented with l-glutamine, 10% fetal calf serum (HyClone, Logan, UT), and penicillin/streptomycin (Mediatech, Manassas, VA). Cultured cells were maintained in 5% CO2 humidified air at 37°C. Constructs expressing GFP fused AKAP-IS and AKAP-IS-Scr were expressed in these cells by transient transfection using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) in Opti-MEM medium (Invitrogen). AKAP-IS and AKAP-IS-Scramble constructs fused with GFP in pcDNA V5/HIS vectors were provided by Dr. John Scott (Alto et al. 2003) and PKA-R1, PKA-RII fused with 6xHIS tag in pET14b vector (Novagen, Madison, WI) were from Dr. Charles Rubin (Dong et al. 1998; Li and Rubin 1995).
Immunoprecipitation, SDS-PAGE and Immunoblot Analyses
Cell proteins were harvested for analysis as described previously (McDonald et al. 1997). Briefly, cells were lysed in ice-cold NDET buffer (150 mm NaCl, 25 mm Tris-HCl [pH 7.5], 5 mm EDTA, 1% NP-40, 0.4% deoxycholic acid, 1 mm NaF, 1 mm Na3VO4 and EDTA-free protease inhibitor cocktail tablets [Roche Pharmaceuticals, Nutley, NJ]) for 1 h with agitation. Lysates were cleared by centrifugation at 13,000 rpm for 10 min. Protein concentration was measured using the BCA method (micro-BCA, Pierce, Rockford, IL).
For immunoprecipitation, cell lysates were precleared with Ultra-Link Protein G-agarose (Pierce) for 30 min at 4°C. Supernatant was then incubated with anti-c-myc (A14G; Santa Cruz Biotechnology, Santa Cruz, CA) or anti-HERG (C-20) antibody for 1–2 h. A nonspecific IgG was used in parallel experiments to control for nonspecific binding. Antibody–antigen complexes were precipitated by protein-G-agarose for 3 h at room temperature or overnight at 4°C. After thorough washing with PBS, proteins were eluted from the resin with 4× Laemmli sample buffer and subjected to SDS-PAGE and immunoblot analysis.
Nitrocellulose membranes were blocked with 10% fat-free milk in Tris-buffered saline (TBS) and incubated with primary antibody solution for 2 h at room temperature or overnight at 4°C. After washing with TBS-T buffer (1 × TBS 0.5% Tween-20), the membrane was incubated with horseradish peroxidase–conjugated (Pierce) or IRDye-conjugated (Rockland, Gilbertsville, PA) secondary antibody in 5% milk TBS-T buffer for 30 min. Immunoblotting results were visualized with ECL or the Odyssey infrared imaging system (Li-Cor Biotechnology, Lincoln, NE). Exposed film (Eastman Kodak, Rochester, NY) was scanned and underwent densitometric analysis. Odyssey software was used to create images and analyze the results.
PKA-Regulatory Subunit Overlay and Far-Western Blotting
PKA-RI and -RII overlay/far-Western was carried out as described by Hausken et al. (1998). Expression of PKA-RI or -RII subunit in pET14b vector (Novagen) was induced by isopropyl β-D-1-thiogalactopyranoside (IPTG) in Escherichia coli BL21(DE3) bacteria. HIS-tagged protein was purified using Ni-NTA resin (Qiagen, Chatsworth, CA). For the overlay assay, cell samples were separated by SDS-PAGE, transferred to nitrocellulose membrane and blocked with 5% nonfat milk and 1% BSA in TBS for 30 min at room temperature. Purified PKA regulatory subunit was added to a final concentration of 0.5 μg/ml in blocking buffer, for a minimum of 3 h at room temperature. PKA-RI and -RII binding was visualized by subsequent Western blot using PKA-regulatory subunit–specific antibodies (BD Transduction Laboratories, San Diego, CA).
Immunofluorescence Analysis and Confocal Microscopy
HEK-HERG cells were plated in glass-bottomed 35-mm dishes (MatTek, Ashland, MA) at low density. Sample preparation was carried out at room temperature. Samples were rinsed with 1 x PBS three times between steps. Cells were fixed with 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) for 15 min and permeabilized with 0.3% Triton X-100 in PBS for 5 min. After 30-min incubation in blocking buffer (5% BSA, 0.1% Triton X-100, 1 × PBS), samples were incubated with primary antibodies diluted in blocking buffer (1:100 for PKA-RI, -RII, and c-myc antibodies from Santa Cruz Biotechnology) for 2 h. Secondary antibodies (Alexa 555 conjugated donkey anti-rabbit, Alexa 647 conjugated donkey anti-mouse antibody; Invitrogen) were reacted with samples at 1:1,000 dilution in blocking buffer for 1 h. After extensive washes, cover glasses were mounted with Gel/Mount (Biomeda, Foster City, CA). Cells were observed using the Leica (Deerfield, IL) AOBS confocal microscope at the Analytical Imaging Facility, Albert Einstein College of Medicine.
In Vitro Phosphorylation
In vitro (or back) phosphorylation was carried out as previously published (Cui et al. 2000). Briefly, HERG protein was isolated from HEK-HERG cells by immunoprecipitation. Antigen–antibody complexes were incubated with purified catalytic subunit of PKA (Sigma, St. Louis, MO) in reaction buffer containing 50 mm Tris–HCl (pH 7.0), 10 mm MgCl2, 0.1 mg/ml BSA and 10 mCi [γ-32P]-ATP for 60 min at 30°C. Proteins were washed with ice-cold NDET, separated by SDS-PAGE and transferred to nitrocellulose membrane. Relative specific activity of HERG-32P was determined by taking the ratio of the scintillation count of the 32P signal over the densitometry of the Western blot signal. To assay for coprecipitation of kinase activity with HERG, immunocomplexes were washed three times and resuspended in 110 μl of kinase reaction buffer with or without addition of 500 nm PKI (PKA inhibitor 6–22 NH2). Next, 5 μCi of [γ-32P] ATP in 10 μl of 880 μm Mg-ATP were added and incubated at 30oC for 30 min. Reactions were analyzed by SDS-PAGE, immunoblot and autoradiography.
Whole-cell patch-clamp current recordings were carried out as previously described (Kagan et al. 2002). Polished patch pipettes with tip resistances of 2–3 MΩ were used. All experiments were carried out at 20–22°C. Cells plated onto glass coverslips the night before were placed into a flow chamber mounted onto the stage of an inverted microscope equipped with epifluorescence illumination and micromanipulators to maneuver patch pipettes. Currents were measured with an Axopatch 200B patch-clamp amplifier (Axon Instruments, Foster City, CA) controlled by a PC using pClamp9 software for data acquisition and analysis (Axon Instruments). Whole-cell capacitance was recorded (generally 10–30 pF) and compensated by analog circuitry of the amplifier. The calculated junction potential for our experimental solutions was 3–4 mV and was not corrected for analyses. The series resistance of 5–12 MΩ (whole cell) was compensated to 75–90% with amplifier circuitry such that the voltage errors for currents of 2 nA were <5 mV. To study I Kr, currents were elicited from a holding potential of −80 mV to depolarizing steps between –70 and +50 mV for 2 s, followed by sequential repolarizing steps to −40 mV and −120 mV. Signals were analog-filtered at 1,000 Hz and sampled at 5,000 Hz. Voltage-dependent activation data were fitted to the Boltzmann relation: I = 1/(1 + exp[(V 1/2 – V)/k]). Current densities were calculated as current (pA) divided by cell capacitance (pF). The voltage dependence of activation was obtained by plotting the normalized peak tail current against the test potential. Internal pipette solution was composed of (in mm) KCl 120, MgCl2 2, CaCl2 0.5, EGTA 5, ATP-Mg4 4, HEPES 10 (pH 7.2, osmolality 280 ± 10 mOsm/L). External solution consisted of (in mm) NaCl 150, CaCl2 1.8, KCl 4, MgCl2 1, glucose 5, HEPES 10 (pH 7.4, osmolality 320 ± 10 mOsm). Data analysis was performed with Clampfit (Axon Instruments) and Origin 7.5 (Microcal, Northampton, MA) software.
Membrane and Microsome Preparation and Velocity Gradient Centrifugation
Fresh porcine cardiac tissue (∼30 g) was homogenized using Polytron PT-2100 (Kinematica, Bohemia, NY) in ∼100 ml ice-cold lysis buffer (10 mm KCl, 2 mm MgCl2, protease inhibitors) supplemented with 0.25 m sucrose. The crude homogenate was cleared with a slow (1,000 × g) and a subsequent fast (10,000 × g) centrifugation (10 min each); the resulting supernatant was then loaded on top of a sucrose cushion (lysis buffer + 0.5 m sucrose) and centrifuged at 34,000 rpm (∼200,000 × g) for 20 min in SW 41Ti rotor (Beckman Instruments, Palo Alto, CA), yielding a membrane-rich pellet ("microsomal fraction"). Velocity gradient centrifugation was then performed using a modification of a previously described method (Okamoto et al. 2001; Sargiacomo et al. 1995). Briefly, either confluent 10-cm culture plates of HEK-HERG cells or pig cardiac microsomes (one-sixth of total material) were lysed in 0.5 ml of MBST (1% Triton X-100 + protease inhibitors in 2-(N-morpholino) ethanesulfonic acid buffered saline (MBS), pH ∼6.5). The entire lysate was loaded atop a 3.7 ml 5–40% linear sucrose gradient (in MBST) and centrifuged at 50,000 rpm (∼340,000 × g) for 16 h in an SW 60 Ti rotor (Beckman Instruments). Liquid fractions (n = 16 or 17) of equal volume were collected from the top, while the pellet was extracted with NDET buffer.
Pattern of AKAP Activity in HEK-293 Cells
AKAP-IS Peptide Affects the Distribution of PKA-RII in HEK-293 Cells
AKAP-IS Decreases PKA-Dependent Phosphorylation of HERG Channels
AKAP-IS Reverses the Acute cAMP/PKA Regulation of HERG Current
HERG Coprecipitates PKA Activity from HEK Cells
Pattern of AKAP Activity and ERG Channels from Porcine Ventricular Myocardium
Acute β-adrenergic regulation of HERG/I Kr may play an important role in the maintenance of cardiac excitability in situations of both normal and maladaptive physiology. That cAMP and PKA are involved in controlling HERG has been shown in vitro (Cui et al. 2000; Thomas et al. 1999) and indirectly from genetic studies (Choe et al. 2006; Schwartz et al. 2001; Wilde et al. 1999). There is evidence, however, that the regulatory system may be more complex in cardiac tissue and may involve additional members of a macromolecular complex (Heath and Terrar 2000; Kagan et al. 2002; Thomas et al. 2003). Here, we provide evidence that that this process may be facilitated by targeting of PKA to the vicinity of the channel by one or more PKA-RII-specific AKAPs. Inhibition of PKA-RII binding to AKAPs in HEK cells with AKAP-IS resulted in altered distribution of PKA-RII, reduced PKA-dependent phosphorylation of HERG protein and reversed PKA-dependent functional effects on the channel.
AKAPs are a structurally diverse group of proteins that bind the regulatory subunits (RII and/or RI) of PKA, thereby targeting and concentrating the enzyme to specific locales of action, generally to specific membrane domains (Colledge and Scott 1999; Rubin 1994). AKAPs often coordinate the scaffolding of various other molecules to a particular site such as protein phosphatases and phosphodiesterase in addition to the specific substrates, resulting in a macromolecular signaling complex (Appert-Collin et al. 2006; Dodge-Kafka et al. 2006; McConnachie et al. 2006). Since the first report of AKAPs (Sarkar et al. 1984), more continue to be added to the list (Feliciello et al. 2001; Ruehr et al. 2004).
PKA-RII regulatory subunit interactions with AKAPs have been the most thoroughly studied; however, RI subunits are also now recognized as targets for AKAPs (Angelo and Rubin 1998; Huang et al. 1997; Li et al. 2001). The AKAPs that have been best established in the heart include mAKAP (also known as AKAP6), AKAP15/18 (AKAP7), Yotiao (AKAP9), AKAP-Lbc (AKAP13), AKAP79 (AKAP5), gravin (AKAP12), AKAP220 (AKAP11), BIG2, MAP2B, AKAP95 (AKAP8), AKAP149/121 (AKAP1) and D-AKAP-1 (AKAP1) (Ruehr et al. 2004). For the most part, these AKAPs target PKA-RII; however, there is evidence for both PKA-RI and RII targeting in the case of D-AKAP-1 and AKAP220. In some instances, the AKAPs have been shown to enhance cAMP/PKA regulation of ion channels in the heart during periods of sympathetic stimulation such as Yotiao and KCNQ1/I Ks (Marx et al. 2002).
Our results suggest that one or more PKA-RII-specific AKAPs may be involved in mediating adrenergic and cAMP regulation of HERG. The AKAP-IS inhibitor is specific to PKA-RII subunits; therefore, we cannot rule out the potential contribution of PKA-RI-specific AKAP targeting to HERG. That ERG channels comigrated with several RI- and RII-binding proteins raises the possibility that more than one macromolecular signaling complex exists for channel regulation. If more than one AKAP could associate with HERG, adrenergic and PKA-mediated modification of channels may differentially occur at various stages of channel synthesis, assembly, trafficking and regulation of I Kr at the surface.
A pressing question that remains is which AKAP(s) is involved in targeting PKA to the HERG channel. Of the known cardiac AKAPs, we have heterologously coexpressed HERG with Yotiao, AKAP220, Ezrin, MAP2, AKAP15/18 and AKAP75/79 with no evidence of coimmunoprecipitation (supplemental data). These results may indicate that other AKAPs are involved or that some intermediary specific to cardiac myocytes is required for association. Alternatively, AKAPs may colocalize to a particular membrane environment (such as a lipid raft) that brings the channel and PKA into a close approximation without direct protein–protein interaction. In the latter case, standard coimmunoprecipitation assays would fail to detect an association even in heterologous cotransfections. That PKA is targeted via Yotiao to the KCNQ1/KCNE1 complex (Marx et al. 2002) was considered, but velocity gradient centrifugation showed no significant difference in sedimentation of RII when HERG was coexpressed with either KCNE1 or KCNE2 (supplemental data). Whether associated KCNE in vivo affects AKAP targeting remains an open question.
These results support further investigation into the nature of macromolecular signaling complexes that may regulate HERG/I Kr. Such studies should give attention to the possibility that different complexes may occur at different temporal and spatial sites of HERG biogenesis and activity. Knowledge of specific signal-transduction entities may impact future medical therapies for various cardiovascular disorders.
We thank Dr. J. D. Scott, Vollum Institute, Portland, OR for providing the AKAP-Scr, AKAP-IS and yotiao constructs and Dr. Charles Rubin, Albert Einstein College of Medicine for providing the AKAP15/18, PKA-RI and PKA-RII constructs and helpful advice on approaches to AKAP investigation. This work was funded by the National Institutes of Health (R01 HL077326 to T. V. M.) and an American Heart Association postdoctoral fellowship (to Y. L.).
Open Access
This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.
Supplementary material
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High-speed local trains: Union Minister Nitin Gadkari's dream project of starting high-speed local trains between Nagpur and four nearby towns namely, Bhandara, Wardha, Katol and Ramtek is soon set to see the light of the day.


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Canopy Bed Wood King
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Karan johar's upcoming movie Takht caught some attention as some sensational Bollywood news
Independence Day 2018; New Railway Timings, Jet Airways scheme and More
The new timetable of Indian Railways is also going to be implemented from the next week on August
know about Delhi Metro restrictions and timings to run till Independence Day celebrations
The Nation is set to celebrate 72nd Independence day on 15th August. Keeping the security measures
Supreme Court denied One State, One Vote Policy in BCCI Case
The Supreme Court on Thursday approved the draft constitution of BCCI with some modifications and
Who is responsible for Fire at BPCL plant in Chembur? 21 people injured
A massive blast and a fire at 2.55 pm happened at the main refinery complex of Bharat Petroleum
Fire breaks out at BPCL RMP Plant in Chembur, Read full story here
A massive blast and a fire at 2.55 pm happened at the main refinery complex of Bharat Petroleum


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June 12, 2018 "At the end of the burst, I felt like we had struck on something vulnerable and real."
Author: Eden Kraus
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BAÑO FLACO CASTAWAY: Our monthly poolside showcase returns this month with a slight twist. Join us at Madame Siam t… twitter.com/i/web/status/1…
.@lilbaby4PF Shows Us Why He's 2018′s Breakout Rap Star
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.@CousinStizz Goes From Hustling to Infamy in 'All Adds Up'
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Back and Seat Chair Cushions Inspirational – Back And Seat Chair Cushions end up being excellent accessories as it offers comfort and style. It is known to highlight the entire interior decoration of a room loaning a warm and cosy sense to your house. There are many types of it used for different kinds of chairs. It is colourful and provide greatest comfort to any kind of seating.
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When you are renting a 5, 10, 15, 20, 30 or 40 yard dumpster, you want a company you can trust with prices that make you smile. Give us a call today and see the difference we can make in your next construction or clean out project.
Simply give us a call and we will help you figure out your dumpster rental needs.
Our dumpsters usually go out same-day or next-day depending on when you call.
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Our trained operators are here to give you a fast and hassle-free experience from start to finish.
Once you are done, simply give us a call and we will get your dumpster picked up right away!
Our staff is here to help whenever you need it. Simply pick up the phone and give us a call.
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Most Merced dumpster rental companies will take care of securing this permit for you if you rent from them. Make sure that if you're planning to put the dumpster on the street, the company has made the appropriate arrangements. You should also ensure that you get the permit in a timely manner and at the correct cost. If you thought the dumpster company was getting a permit and they did not, you will be the one who will have to pay the fine that is issued by the authorities.
You will need a dumpster to throw out the paneling and outdated carpeting with. You will need to toss the old doorways as well if you're replacing these people.
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Please use the form below to contact us. We look forward to working with you and will get back to you as soon as possible. All fields are required


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Grizzlies vs. Kings recap - GCM
MEMvSAC SL Playlist - 7/9/17
Grizzlies vs. Kings recap - GCM
Grizz-Mo: Grizzlies vs. Kings
Grizzlies vs. Kings highlights
Wade Baldwin on win over Kings
Davis for two
Assist by Selden
Martin's two-handed slam
Summer League: Coach Cyp on prep for MEMvSAC
Wayne Selden Postgame Interview
Pete's Perspective: Grizzlies earn 81-75 win over Kings
It wasn't a thing of beauty, but the Memphis Grizzlies pushed their record to 2-0 in Summer league play with a gritty (what else?) win over the Sacramento Kings by an 81-75 count inside Cox Pavilion.
The Grizzlies took control of the scoreboard in the second quarter, outscoring the Kings 24-17 in the frame. In that period, the Grizzlies hit 10 of 18 shots and dominated on the glass against the cold-shooting Kings.
Still, the Grizzlies nearly blew a 17-point lead, much as they did a 16-point advantage Saturday against Washington. But Wayne Selden and Jarrell Martin rose to the occasion. Those two Grizz posted 17 of the team's 21 points. Clinging to a 71-68 advantage with just under three minutes remaining, Selden connected on two free throws, then saw Martin slam back a Baldwin miss and next assisted on another Martin slam off a gorgeous press-break. That picture-perfect execution slammed the door on the Kings.
Sacramento got 17 points and 4 assists from heralded rookie De'Aaron Fox, but he had more turnovers than assists and more fouls than either category. Justin Jackson's shot went MIA as he clanked all eight attempts from deep, finishing with four points.
Quick takeaways from the game:
- Nice bounce back game for Jarrell Martin. After a less-than-stellar outing against Washington, Jarrell showed why Memphis drafted him in the first place: perimeter shooting and inside presence. His 21-point, 10-rebound effort was a definite upgrade.
- Wayne Selden played strong, assertive basketball and if this continues, he'll be impossible to keep out of the rotation. He filled up the stat sheet in all the right columns and was a gamer when Sacramento made their run.
- Wade Baldwin was much better in game two. He rebounded the ball well (ten boards) and scored passably well. He also turned it over just once. He also accepted the challenge of dealing with lottery pick De'Aaron Fox. Now, he needs to string strong performances together.
- Rade Zagorac looked much more comfortable in posting six points and five rebounds.
- After a strong performance against Washington, Kobi Simmons came back to earth with a team-worst -13. In fact, the entire Grizzlies bench was in negative territory and was part of the reason Memphis nearly gave away the lead.
Overall, the Grizzlies played with greater force and precision than Sacramento. The Kings bricked shot after shot and if not for the rebounding efforts of Skal Labissiere and Jack Cooley, the Grizzlies would have won comfortably.
The vibe around Sunday night's Summer League matchup between the Grizzlies and Kings was an odd combination of family reunion and family feudin'.
The most beloved played to wear Beale Street Blue, Zach Randolph, is now a King and the most decorated player to wear the bear, Vince Carter, will be his teammate in the California capital. The only constant is change, but this was a stunner for Grizzlies fans.
There is no quick and concise way to summarize Z-Bo's impact in Memphis, except for this: Z'Bo is Memphis. He understands having to scrap and scrape and fight from the bottom up. There's no glitter on his game, just grit and grind. And despite the scowls (he's looking at you, Steven Adams) and shoves (hi there, Blake Griffin), he's smiles and sincerity to his people.
I've known Zach since he was a rookie in Portland. His transition to the NBA wasn't easy. He was young, impressionable and socially out of his element in Portland. Surrounded by questionable associates, he developed a less-than-sterling reputation. But Zach disproved F. Scott Fitzgerald's belief about there being no second acts in American lives.
When Michael Heisley signed Zach to the Grizzlies, he made it clear that Zach's tenure with the Grizzlies was related to his comportment. A handful of NBA Community Assist awards, thousands of dollars donated and smiles generated later the larger world has discovered what Zach Randolph has always been about – being a big-hearted and loyal man.
And if the world had more Vince Carters, we'd all be better off. Take away the dunking, the three-point shooting and the "rev it up" celebrations and you still have a man in full. Thoughtful, engaging and earnest in his desire to leave the world a better place, Vince Carter is a big believer in education. Just ask Grizzlies Prep and its students and staff.
Forget about basketball for a second. If Vince Carter had gone into business, he'd be the CEO by now. He's a natural leader and loves being among people. After what would turn out to be his final Grizzlies exit interview, he came to the practice facility to meet the media. Unlike most players, Vince didn't immediately depart after those responsibilities. Instead, he stayed and visited with a number of Grizzlies staffers who were just hanging out, drinking in the last of a season just ended. Five minutes, then ten minutes stretched to twenty or more. Vince stayed. He chatted, laughed and reminisced.
If all you know of Vince Carter is dunking, you only know maybe 10% of the man. Memphis was privileged to know 100%.
Next Game
Grizzlies tangle with the Utah Jazz on Tuesday, July 11 in Game 3 of the Las Vegas Summer League at 3:30 p.m. CT. Catch all the action on NBATV.
Key Stat of the Night
- The Grizzlies outshot Sacramento from the charity stripe by a 71.9 percent to 57.1 percent margin.


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Holy Cross Church was established in the 12th century and has been maintained through the years. Several points of interest can be found in the church and the churchyard cemetery. The Viscount Mont of Alamein's grave can be found in the far northwest corner. His headstone lies flat and is poignant in simplicity. Less easy to find and hidden away in the choir vestry is the effigy of a Crusader Knight who may have fought in the 7th Crusade under King Edward I. It has the inscription: "Richard de Westcote gist ici deu de sa alme cit merci amen" presumably the monument of the father or grandfather of Richard de la Bere. Overlooking a village and farmland, the church with its red tile roof is situated on a mount with a red brick wall surrounding it and the cemetery. The church is constructed of the underlying rock, called malm stone, a mixture of a chalky marl and chiloritc grain like mica, which is similar to the character of limestone.
GPS Coordinates: 51.16253, -0.89816
- Added: 22 Jul 2001
- Find A Grave Cemetery: #638815
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How to Choose an Exterior Home Color Palette
Ideal email subject line: Would you paint your house black? See what happens when you do...
**You can also write your own subject line. Just be sure to avoid using exclamation points or all caps, and be sure the subject is attention-grabbing.
Ideal sending date / time: Any Tuesday, Wednesday, or Thursday between 7 AM and 1 PM (or try 5 or 6 PM to see if your open rates increase!)
Goal of this newsletter: Help your potential clients understand how difficult it can be to put together a cohesive exterior color palette for their homes. This newsletter sets you up as the professional who can make color choices easy for them.


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Singing Pianist / Keyboard Player
Kingston, ON
33 miles from Watertown
Sharon Wickett is a versatile and creative performer. Over the years, she has travelled from coast to coast…
Philip Fillion, Organist and Pianist(1)
Organist / Keyboard Player
Auburn, NY
79 miles from Watertown
Recent review: Mr. Fillion was the organ accompanist for the Faure Requiem, which was performed at the Church…
Mac&Keys - Cover Band (Keyboard/Vocals) and DJ(1)
Keyboard Player
Ottawa, ON
100 miles from Watertown
Recent review: Our event had 60-70 people ranging from 2-70years old. Chris put on an incredible show, with…
Nicholas Deek - Pianist(0)
Pianist / Keyboard Player
Ottawa, ON
100 miles from Watertown
My goal is to make the music the most or one of the most special parts of your occasion. I perform large to…
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Singing Pianist / Keyboard Player
Rochester, NY
101 miles from Watertown
I'm a graduate of SUNY Onondaga Community College with my Associate's in Music, currently attending Nazareth…
Arthur Ward(9)
Singing Guitarist / Keyboard Player
North Tonawanda, NY
161 miles from Watertown
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Buffalo, NY
167 miles from Watertown
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Toronto, ON
175 miles from Watertown
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Toronto, ON
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Watertown NY Keyboard Players
Keyboard players are a perfect choice for events of all types from corporate and community events, to birthday parties and weddings. They can provide a variety of sounds and styles of music. Electronic keyboards can synthesize unique sounds and multiple instruments, making it a great substitute for a full sized band. Keyboard players can provide a similar service as a piano player, but offer the convenience of portability that you can't get with a baby grand or upright piano. Search for Keyboard Players in Watertown, New York above.
Please note that these Keyboard Players may also travel to Glen Park, Brownville, Calcium, Black River, Limerick, Dexter, Rodman, Evans Mills, Felts Mills, Fort Drum, Adams Center, Sackets Harbor, Great Bend, Deferiet, Chaumont, Adams, Copenhagen, Depauville, Carthage, Lorraine, La Fargeville, Philadelphia, Henderson Harbor, Deer River, Belleville, Denmark, Theresa, Pierrepont Manor, Henderson, and Three Mile Bay.


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When you are renting a 5, 10, 15, 20, 30 or 40 yard dumpster, you want a company you can trust with prices that make you smile. Give us a call today and see the difference we can make in your next construction or clean out project.
Simply give us a call and we will help you figure out your dumpster rental needs.
Our dumpsters usually go out same-day or next-day depending on when you call.
We provide top-notch service, while going easy on your bottom line. What more could you ask for?
Our trained operators are here to give you a fast and hassle-free experience from start to finish.
Once you are done, simply give us a call and we will get your dumpster picked up right away!
Our staff is here to help whenever you need it. Simply pick up the phone and give us a call.
Most cities or municipalities do not have many regulations regarding dumpster rental as long as you keep the dumpster entirely on your own property during the rental period. If you need to place your container on the street in any way, you will likely have to obtain a permit from the appropriate building permit office in your town. You can check with your local public works department to be sure.
Most Brooklyn dumpster rental companies will take care of securing this permit for you if you rent from them. Make sure that if you're planning to put the dumpster on the street, the company has made the appropriate arrangements. You should also ensure that you get the permit in a timely manner and at the correct cost. If you thought the dumpster company was getting a permit and they did not, you will be the one who will have to pay the fine that is issued by the authorities.
Make sure you learn how long you can preserve the rented dumpster. You should be equipped to use it for one particular fee for around two weeks. Find out you can rent the idea a little longer if your job is large and you don't think the contractor will conclude within this time.
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Any type of rubbish you are throwing in will also establish the rental company you use. When the material you need to throw out is definitely hazardous, you may need to make sure the company can get rid of it effectively.
There are quantity of 6 Yard Dumpster in Brooklyn, PA companies in the marketplace who can assist you in this consider. This is the most economical way of wiping out huge a great deal of piled up waste in the smallest time feasible. Dumpster rental companies distributed across the marketplace will be designed with wide verities connected with dumpsters to suit your unique requirements, yet a throw off dumpster will certainly be a best option to get rid of the trash about community cleaning.
An ideal company to rent from is certainly one that offers an appartment rental rate. The actual charge will incorporate delivery, pick-up, disposal in addition to taxes may also be included. Many companies in addition only have a four as well as five-day rental period, and all days past these times frame are an additional impose. Renting from a company with a longer rental period is much more desirable.
Hire a contractor to obtain the remodeling accomplished. He or she can change your house windows, baseboards, sheetrock, carpeting, vanities, units, old hardwood flooring, etc. The dumpster will come in useful when hurling all of the older items available.
Are you cutting down trees on your lawn or have a lot of older car pieces and crap laying around your own yard? You can actually pick up your own yard whenever you rent a following day dumpster. Enjoy having a brand new and thoroughly clean yard when you finish up the job. You will really feel a whole lot far better knowing how wonderful your property looks just as before.
Now you know what size dumpster you will need when renovating a building, try to find wheels for the dumpster you need to rent as well as a company you are able to request skids to protect your front yard.
You may be undertaking extensive focus on your bathroom in addition to plan on shredding out the porcelain tile in your restroom or having a person else do it. The person carrying it out will be able to effortlessly throw the older tiles in addition to old shattered tiles available safely in the dumpster.
Roll down dumpsters usually arrive in large sizes ranging from 10 property to 40 property and are cable television of dealing with huge a great deal of trash with one get. This will help keep you away from acquiring multiple journeys to dispose the particular waste stacked up on your local community cleanup. This will likely not only conserve much of your time but also help make your trash convenience task affordable. Dumping the particular bulky waste into the dumpster painless when you employ a roll down dumpster for your waste disposal undertaking because a throw off rented from the 6 Yard Dumpster in Brooklyn, PA companies is specially designed to make it a finest fit pertaining to bulky waste pickup. These special waste bins are mounted on small wheels so that they can easily be scrolled on to the particular trucks ready to carry these people to the landfills. They are closed with the increase doors one particular on the top and something at the bottom, the door at the bottom may facilitate uncomplicated dumping on the bulky waste and door on the top is definitely open pertaining to disposal on the light weight.
Today, how to approach hiring a specialized 6 Yard Dumpster in Brooklyn service provider? Before you go hire one of the professional carrier's networks, one should possess transparent snapshot about the quantity of the throw away that is going to be got rid of and whether the 6 Yard Dumpster in Brooklyn service provider can accommodate your requirements. you need to go through the commitment agreement from the Dumpster rental service provider as well as familiarize yourself with this terms and conditions from the agreement because there are limits without a doubt companies within the quantity throw away as well as objects disposed from the waste ensure that you fit into the criteria. It is always wise to have a earlier check with service agency on all these issues before you are going hire a single.
You can order this bin or dumpster according to the need. These come in different sizes as well as shapes. In accordance to the quantity of the trash, you can place needing the same.
Speedy service including next day 6 Yard Dumpster in Brooklyn is the one other fantastic product to look for. If you are in a hurry to receive the project started out and finished, this is a will need to have option.
You will need time and operate to take out the bathroom ., vanity, disparaging offer out the flooring of your restroom and grab the baseboards off. Once you have finished this you'll be able to throw the debris into the next day 6 Yard Dumpster in Brooklyn you have soaking in your yard. Quickly throw the debris absent and simply remove the mess once the dumpster is gone from a yard.
Still 6 Yard Dumpster in Brooklyns aren't one size fits all. To enable you to keep your fees in check, you have to pay only for this receptacle sizing that suits the amount of rubbish. So whether or not you rent a 10-, 20-, or 30-yard capacity dumpster, you'll have precisely the expanse regarding space you may need to jettison all of your useless. Contact your local junk hauling service currently for a free of charge, no-obligation estimate within the different sizes regarding dumpsters.
If you are a enterprise you are probably investigating having the garbage 6 Yard Dumpster in Brooklyn consistently. But if you are doing a project with a home, you happen to be looking at a set fee of time to contain the unit. Commonly for these kinds of projects, you will need the discretion unit with regard to anywhere from a few days to a few weeks. Whilst businesses probably have to enter into binding agreement that will finally save these people money, house users might not. This is where watchful research is needed. While a company can possibly negotiate a lower rate if they can guarantee usage for a set period of time, smaller projects might not be ready to do so. Bring your search on the internet to help you be sure to get the best deal for you are tapering to do.
You can easily get rid of the garbage or waste in which builds up every day when you rent a dumpster. By renting a dumpster you have made your lifestyle that much much easier versus seeking to get rid of the many garbage with a few loads via truck. A dumpster can help you speed up particles the house getting built. You simply won't have to enable time and petrol money moving the throw away of vacant packaging as well as boxes back to this employers.
You should also check if there are virtually any environmental costs and fees intended for distance included to get rid of the rubbish because has an effect on how much the service charges.
Dumpster diving is an easy way to get extra money. It is like a treasure hunt but you are surrounded by waste. You can easily discover valuable products and sell these people without any cost to you. Look at the local regulations and give this a shot.
Dumpster rental fees can assistance homeowners, businesses, churches, agents, and group effort assignments. The 6 Yard Dumpster in Brooklyn, PA procedure is very easy and simple, and affordable. There are various measured Maryland dumpster rental fees available with a medium sized dumpster together with wheels upon it. Next day supply options make cleanup simple and efficient. Neighborhood trash pick up regulations minimize the amount of trash that is available intended for pickup by the city. Maintain your home as neat and clean as it can be during your your kitchen renovations by using a development 6 Yard Dumpster in Brooklyn, PA.
It is also important to find out how much the company will charge you when you decide to employ a rubbish receptacle. Now and again, there are added charges when you exceed some limit with regards to the rubbish you put in to the receptacle. That is something that you need to find out could you signal a contract that has a local company. It is far better to choose a company that has its own landfill. If the company doesn't need its own, many of them have to pay to train on a landfill in your community and this charges will definitely always be passed for you.
Dealing with surplus garbage, whether or not it's from clearing out an real estate or cleaning up after development demolition, can be an arduous undertaking. Historically, the way in which people have accumulated and gotten rid of debris like this was to rent a dumpster and have the trash picked up that way. Today, however, there's a brand-new option on the market: the Big Tote. The Big Tote is, since the name shows, a large, sturdy bag that can be filled up with still types of trash that would normally go in a dumpster. The rubbish is then collected using a junk treatment service, making your property clean up of all which trash for good. The Big Tote junk treatment option is a most wonderful choice for almost just about every trash hauling job and beats out dumpster-style bins in terms of cost and convenience almost every time.
Finding a reputable and affordable 6 Yard Dumpster in Brooklyn, PA company really should not be too hard to perform. By following uncomplicated guidelines, you need to be able to solicit the services of a company that will attend to your needs in a timely fashion and provide you with some sort of satisfaction assure policy. Understand that going with a local company versus using a national you are going to provide you with a larger chance of discovering dependable assistance.
Next you can put your newly stained baseboards to the lower wall membrane and floorboards. If you requested new opportunities and you or the door corporation have stained them, the next step is to put these people on their hinges.
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Cat Tree Bruno - beige
The cat tree Bruno offers a wide variety of opportunities for jumping climbing and relaxing. Of course there´s plenty of space to scratch too! This lovely cat tree fits into nearly any apartments décor and offers your pet plenty to do without taking up much floor space. Dimensions Base 60 x 60 cm Barrel/Den 61 cm ø 37 cm 2 entrances Posts 1 x 60 cm 3 x 40 cm 1 x 39 cm 2 Platforms 40 x 60 cm 35 x 35 cm Hammock ø 45 cm 5 sisal posts each 9 cm This product cannot be delivered to Northern Ireland and Switzerland.
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Sisal Cat Scratching Board - 70 x 17cm
This scratching board can be positioned easily at any appropriate height. Dimensions 60cm x 11cm - sisal pad 55 cm x 11 cm 70cm x 17cm - sisal pad 62 cm x 15.5 cm Cats need to sharpen their claws regularly - the trick is to keep them from using your furniture for the job! Providing a suitably hard-wearing material is vital; the tough natural fibres of sisal are ideal helping your cat to shed dead layers of nail and preventing ingrown claws. Please note Colour of the Scratching board may vary from the product image.
Trixie Turbinio Cat Toy - 1 toy
Turbinio is the ideal Trixie toy to keep your cat occupied. Even if you haven´t time to play with your soft-pawed friend Turbinio will keep boredom at bay! This cat toy animates your cat with its automatic movements that keep pulling the mouse on the cord. Your cat will just keep playing and playing! The ball is made from plastic and includes a battery-run motor. Please Note As with any other product your pet should be supervised with this toy. Please check the product regularly for any damage and replace the toy whenever it becomes defective or when a piece of the toy goes missing to avoid any harm coming to your pet. What´s this?
Day & Night Cat Ball Toy - 2 items
These balls are just like little ghosts. They glow in the dark and keep your cat spellbound. The bells and furry tails will entice your cat to play crazy games of hunt and chase. Give your cat a ghostly surprise with this unusual cat toy. Diameter 4 cm Please note As with any other product please supervise your pet with this toy. Check the product regularly for damage and replace the toy if it is damaged or missing any pieces to avoid potential injury to your pet.
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In this study, both solid-phase and fluid-phase temperatures inside a porous cathode of a polymer electrolyte fuel cell in contact with an interdigitated gas distributor are predicted numerically. The porous cathode consists of a catalyst layer and a diffusion layer. The heat transfer in the catalyst layer is coupled with species transports via a macroscopic electrochemical model. On the other hand, in the diffusion layer, the energy equations based on the local thermal non-equilibrium (LTNE) are derived to resolve the temperature difference between the solid phase and the fluid phase. As for the species transports, the Bruggemann model is employed to describe the effective diffusivities of the oxygen and water vapor in the porous cathode. Results show that the wall temperature decreases with increasing the intrinsic heat transfer coefficient. As the intrinsic heat transfer coefficients increase, the porous electrode becomes local thermal equilibrium with a strong thermal interaction (heat transfer) between the solid and fluid phases. Under the conditions of high intrinsic heat transfer coefficients, the temperature difference between the solid matrices and the reactant fluids are negligible. © 2006 Elsevier B.V. All rights reserved.
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Alexandria, VA Dowell Dowell
YP - The Real Yellow PagesSM - helps you find the right local businesses to meet your specific needs. Search results are sorted by a combination of factors to give you a set of choices in response to your search criteria. These factors are similar to those you might use to determine which business to select from a local Yellow Pages directory, including proximity to where you are searching, expertise in the specific services or products you need, and comprehensive business information to help evaluate a business's suitability for you. "Preferred" listings, or those with featured website buttons, indicate YP advertisers who directly provide information about their businesses to help consumers make more informed buying decisions. YP advertisers receive higher placement in the default ordering of search results and may appear in sponsored listings on the top, side, or bottom of the search results page.
1. Dowell & Dowell
2560 Huntington AveAlexandria, VA 22303
2. Dowell Enterprises Inc
8605 Hillside PlFairfax, VA 22031
3. Garrett, MacDowell I Atty
437 N Lee StAlexandria, VA 22314
4. Michael Clay McDowell
4660 Kenmore Ave Ste 900Alexandria, VA 22304
5. McDowell, Heather L
575 7th St NW Lbby 1Washington, DC 20004
6. McDowell Suzanne Ross
1330 Connecticut Ave NW Ste 1CWashington, DC 20036
7. McDowell, Robert L, MD
4821 Fort Sumner DrBethesda, MD 20816
8. McDowell, Claudia T
8270 Willow Oaks Corporate DrFairfax, VA 22031
9. Dowell Associates
8700 Edgeworth DrCapitol Heights, MD 20743
10. Dowell Associates
8800 Edgeworth DrCapitol Heights, MD 20743
11. McDowell Jr, Robert L, MD
Dr. McDowell is very knowledgeable on all issues regarding kids and their development. Also, my kids really enjoy his sense of humor and his abilit…
12. MacDowell & Associates, P.C.
4031 University Dr Ste 500Fairfax, VA 22030
From Business: MacDowell & Associates, P.C, have Attorney who have excellent experiance in the area of drunk driving and traffic violations
13. McDowell, Kara
4301 Jones Bridge RdBethesda, MD 20814
14. Law Office Jaimee C McDowell
11885 Holly LnWaldorf, MD 20601
15. Rosenzweig & McDowell
6550 Rock Spring DrBethesda, MD 20817
16. Vesta D McDowell, DPM
7425 Forbes Blvd Ste 105Lanham, MD 20706
17. Helen O McDowell, LCSW
107a W Edmonston DrRockville, MD 20852
18. Wilson Dowell Farms
2905 Chaneyville RdOwings, MD 20736
19. Dowell Financial Service
140 Dalrymple RdSunderland, MD 20689
20. Mc Dowell & Mc Dowell
11705 Lord Philip CtGermantown, MD 20876
21. Rossi Kimms & McDowell LLP
20609 Gordon Park Sq Ste 150Ashburn, VA 20147
22. McDowell, Aimee, RN
10440 Little Patuxent PkwyColumbia, MD 21044
23. McDowell, Kenneth, MD
392 Garrisonville RdStafford, VA 22554
24. Dorian C Dowell
5537 Windysun CtColumbia, MD 21045
25. Shirnia & Dowell - CLOSED
9818 Perrott CtFairfax, VA 22031
26. Dowell Farms - CLOSED
PO Box 453Sunderland, MD 20689
27. Dolley Madison Towers
2300 24th Rd SArlington, VA 22206
From Business: Dolley Madison Towers is located right next to the Army-Navy golf course, giving our residents a beautiful overlook. These residences are just one block from the …
28. Dominion Towel Supply
9665 Gunston RdFort Belvoir, VA 22060
We found28results


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Thank you for your inquiry about employment opportunities with NAMIC. Currently we do not have any position openings available.
NAMIC again has been named as one of the 100 Best Places to Work in the Hoosier State for 2017 by the Indiana Chamber of Commerce, earning the number three ranking in the category of medium-sized employers with between 75 and 249 employees.
Chuck Chamness, president and CEO, cites NAMIC's corporate culture that is professional yet friendly as the reason for the association's consistent showing on the Best Places to Work list. "NAMIC member companies recognize and appreciate the meaningful work done by our dedicated staff. Knowing that their efforts are appreciated, while working in a collaborative team environment alongside great co-workers, is what sets our work environment apart for staff. The added perks that support a positive work atmosphere just reinforce the overall culture of the organization."
The 2017 rankings represent NAMIC's fourth selection to the Best Places to Work in Indiana list, and the third time that NAMIC has been ranked in the top ten companies for its size category, and the second time in the top five.