to the number of GLR-1::GFP pixels under threshold. To maximize our resolving power while observing the relatively small C. elegans neuron cell bodies, we restricted our analysis to a single focal plane taken through the middle of each cell body. As an alternative approach, we also analyzed the same images using a confined displacement algorithm run in ImageJ as previously described [79]. Manders colocalization coefficients were deter- mined for the real data and for the randomized image, and then compared statistically to obtain p values. Behavioral Assays The reversal frequency of individual animals was assayed as previously described, but with some modifications [51]. Single young adult hermaphrodites were placed on NGM plates in the absence of food. The animals were allowed to adjust to the plates for 5 minutes, and the number of spontaneous reversals for each animal was counted over a 5-minute period. Twenty animals were tested for each genotype, and the reported scores reflect the mean number of reversals per minute. GST Pull Downs Complete sequences for ubc-13 were introduced into the GST expression vector pGEX-GW (a gift from Barth Grant), and either GST alone or GST::UBC-13 were expressed in E. coli strain BL21 and purified using glutathione-Sepharose as described previously [97]. Complete sequences for uev-1 or uev-1 mutated F8A were introduced into the pcDNA3.1-GFP expression vector (a gift from Barth Grant), and transfected in COS7 cells for 48 hours. Cell were harvested and lysed in TEEN (20 mM Tris, 1 mM EDTA, 1mM EGTA, 100 mM NaCl) plus 1 mM PMSF, and Triton X-100 was added to 1% for 1 hour at 4uC after needle aspiration. For each experiment, 12 micrograms of GST protein was added to 400 microliters of solubilized supernatant, incubated for 45–60 minutes at 4uC, and washed several times in TEEN plus 0.2% Triton X-100. Proteins were eluted into loading buffer and separated by SDS- PAGE for Western blot analysis using anti-GFP antibodies. Yeast Two-hybrid Interactions Yeast two-hybrid experiments were performed by placing ubc-13 and uev-1 cDNA sequences into the pEG202 bait vector and pJG4-5 prey vector. The resulting plasmids were cotransformed, along with the reporter plasmid pSH18-34, into yeast strain EGY48, and transformed yeast were recovered on –His –Trp –Ura dropout plates. Resulting colonies were diluted in series on –Leu –His –Trp dropout plates to test for interactions based on growth. K63-linked Anti-ubiquitin Western Blotting Western blots with Apu3.A8 were performed essentially as described previously with modifications [90]. Nematodes were dounce homogenized, needle aspirated, and sonicated in 20 mM Tris (pH 7.4), 1 mM EGTA, 10% glycerol, 135 mM NaCl, 1.5 mM MgCl2, 1% Triton X-100, 6M urea, 2 mM N- ethylmaleimide, 25 micromolar MG132, and a protease inhibitor cocktail (Roche). Total protein was quantified by Bradford assay, and then SDS was added to a final concentration of 1%. The lysates was centrifuged at 13,000xg to remove insoluble material, and equal quantities of extracted proteins were separated on SDS- PAGE gels. After electrophoresis, resolved proteins were trans- ferred to PVDF membranes (Immobilon-P) and blocked with 2% BSA in PBS with Triton X-100. Membranes were incubated with Apu3.A8 anti-K63-linked ubiquitin antibodies (Millipore) at 1:300 for 1 hour, or C4 monoclonal anti-actin antibodies (MPBiomedic) at 1:1000 overnight at 4uC. After washing, HRP-conjugated UEV-1 and Glutamate Receptors secondary antibody was applied at 1:2000 for 1 hour at room temperature. Immunoreactive bands were visualized using the enhanced chemiluminescence (ECL) system (GE Healthcare). GLR-1::GFP Western Blotting Lysates were prepared from adult worms using a Wheaton DuraGrind stainless steel dounce homogenizer and buffer A (50 mM Hepes pH 7.7, 50 mM potassium acetate, 2 mM magne- sium, 1mM EDTA, 250 mM sucrose), a protease inhibitor cocktail (Roche), and 10 mM N-ethylmaleimide. Membranes were isolated from clarified lysates by ultracentrifugation, and then suspended in buffer A plus b-mercaptoethanol, SDS, and DTT. Proteins were separated from membrane lysates by SDS-PAGE, and GLR- 1::GFP or actin were simultaneously detected by Western blotting using a combination of anti-GFP antibodies (GeneTex Inc.) and anti-actin antibodies (MP Biomedicals). Secondary antibodies conjugated to infrared-emitting tags were used to detect both primaries on the blot using an Odyssey LI-COR. Quantitation was performed by comparing samples to a standard curve generated by diluting samples from wild-type lysates. Real-time PCR Total RNA from adult worms was prepared using Trizol (Invitrogen). First strand reverse transcription was performed using iScript (Biorad) as per the manufacturer’s instructions. Real-time PCR reactions were performed using iQ SYBR Green (Biorad) and a BioRad real-time PCR cycler as previously described [63]. Primer sets detected either glr-1 mRNA or mRNA for dlg-1, a control transcript of an adherens junction protein [98]. Primers spanned intron junctions so as not to detect genomic DNA, and no product was detected in the absence of reverse transcription. Standard curves were generated and analyzed in triplicate by dilution. Concentrations were derived by comparing Ct values of samples to standard curves. For each genotype, mean glr-1 expression was normalized to that of dlg-1. Supporting Information Figure S1 The uev-1 transcription unit. Genomic sequences in and around the uev-1 transcription unit, from the start of transcription until the final nucleotide present in the mRNA, are shown. Capital letters highlighted in gray indicate coding sequences within exons. Numbers are based on nucleotides starting from the ATG and containing only exonic sequences. The horizontal bar indicates sequences missing in the ok2610 deletion. The nonsense mutation in od10 is also indicated. Found at: doi:10.1371/journal.pone.0014291.s001 (2.68 MB TIF) Figure S2 Overview of GLR-1-Expressing Neurons. Top panel diagram indicates the position of the pharynx (green), command interneuron cell bodies (red circles, only left side shown for clarity), RIG cell bodies (blue circles), AVG cell body (purple circle), and the various neurite projections into the nerve ring and along the ventral cord in the head region. The PVC command neuron cell body, which is located in the tail, is not show; however, the PVC neurite would belong to the bundle of fibers along the ventral cord and projecting into the nerve ring as indicated in red. Note that the RIG neurites do not enter the ventral cord until the most anterior portion of the lateral ganglion; thus, no RIG synapses are included in our analysis [100]. Also note that AVG makes only a single synapse in the retrovesicular ganglion region of the ventral cord; thus, the contribution of AVG to our analysis is minimal [100]. Bottom panel diagram indicates the position of cell bodies and neurites from a dorsal view. The circular nerve ring has been flattened out to lie in the same plane as the ventral cord so that the PLoS ONE | www.plosone.org 18 December 2010 | Volume 5 | Issue 12 | e14291