Fig. 6: LIN-2 binds to the TM3-4 loop of UNC-49 and to NLG-1.
a Domain composition of NLG-1 and LIN-2B. b LIN-2B binds NLG-1 intracellular domain (NLG-1_ICD) in yeast two-hybrid assays. By contrast, LIN-2B does not bind NLG-1 after deletion of the PDZ binding motif (NLG-1_ICD∆PDZB). c, d Confocal detection of RFP-labeled GABAARs in nlg-1(0) null mutants expressing full length NLG-1-GFP or NLG-1_∆PDZB-GFP in muscle. GABAAR fluorescence levels were normalized to the wild type. Box plot as in Fig. 1e. Two-tailed Student’s t test, ***p < 0.001. e Direct binding of GST-LIN-2B and HA-UNC-49B_TM3-4 intracellular loop was detected in GST pull down assay. The samples were analyzed by immunoblotting using an anti-HA antibody. The same membrane was stained with an anti-GST antibody to show GST expression. The experiment was repeated twice. f Pull down experiment using GST-tagged UNC-49B_loop and HA-tagged full-length and deleted versions of LIN-2B. Samples were analyzed by immunoblotting using an anti-HA antibody. The same membrane was stained by ponceau S red to show GST expression. g, h Co-immunoprecipitation from extracts of wild-type and unc-40(0), lin-2(0) and frm-3(0) null mutants expressing RFP-UNC-49 and NLG-1-GFP in muscle. Worm lysates were precipitated with anti-GFP nanobody-coupled TrapA beads. Samples were analyzed by western blot using anti-RFP or GFP antibodies. i, j RFP band intensities were normalized to GFP bands. Data are presented as RFP/GFP ratios normalized to the wild type. Box plots are as in Fig. 1g. Two-tailed Student’s t test ***p < 0.001, **p < 0.01. The experiment was repeated three times. Scale bar = 10 μm.
