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. 2020 Oct 8;29:95–106. doi: 10.1016/j.jare.2020.09.009

Fig. 4.

Fig. 4

Interaction between collybistin and the GlyRα1 TM3-4 loop. (A) Schematic structure of collybistin variants 1–3. (B) Binding studies between α1-GlyR and collybistin I-II using GST pull down assay upon HEK293 cell transfection with CB1SH+-HA and CB2SH--HA. GST-fusion proteins of the isolated GlyRα1 wildtype or GlyRα1P1-5A TM3-4 loop were recombinantly expressed in E. coli BL21 cells, coupled to glutathione-sepharose beads and finally incubated with HEK293 cell lysates. Results are shown after Western Blot analysis. Lane 1: input, lane 2: GST control, lane 3: α1-wt TM3-4 loop, lane 4: GlyRα1P1-5A TM3-4 loop. C Co-immunoprecipitation of glycine receptors and collybistin splice variants. The GlyRα1-specific antibody mAb2b was used for co-precipitation. Lane 1: GlyRα1 + CB1 (1:1); lane 2: GlyRα1 + CB2 (1:1); lane 3: GlyRα1 + GlyRβ + CB2 + gephyrin (1:2:2:2); lane 4: untransfected cells; lane 5: GFP = mock control (left panel). Collybistin was detected at the appropriate molecular weight of 60 kDa (precipitated with GlyRα1 – see IP, upper panel and input control expression of collybistin - see input second panel; gephyrin at 93 kDa, and GAP-DH at 32 kDa. The observed shift in molecular weight between CB1 and CB2 is due to the presence of the SH3 domain in CB1 but not CB2. Right panel: Quantification of the relative collybistin expression normalized to GAPDH. At least 4 independent experiments have been performed and were used for quantification analysis. (D) Co-immunoprecipitation of GFP-CB2 or GFP-PH domain together with pCIS-HA-GlyRα1. Beads were coupled with mouse anti-HA antibody or mouse IgG, HEK293 cell lysates were incubated with antibody coupled beads (see methods). Samples were subjected to SDS PAGE and Western Blotting. Lane 1: input; lane 2: IgG control; lane 3: IP HA tag. Left panel: GFP-CB2; right panel: GFP-PH domain. (E) Electrophysiological data after co-expression of GlyRα1 with CB1 or CB2. Left panel: EC50 curve of GlyRα1 (solid squares, solid line); GlyRα1 co-expressed with CB1 (open circles, dashed line) and GlyRα1 co-expressed with CB2 (open triangle, dotted line). Right panel: comparison of GlyRα1 and co-transfection with CB1 and CB2. Differences were not significant (p > 0.05, one-way ANOVA).