Figure 2.

The GABA_AR α5 subunit is able to directly associate with gephyrin. (A) Cultured hippocampal neurons were transfected with α5^WT and lysates were immunoprecipitated with anti-GFP or anti-IgG. Precipitates were immunoblotted for GFP and gephyrin. Immunoprecipitation of GFP resulted in coimmunoprecipitation of gephyrin. (B) Cortical and hippocampal (HPC) lysates from adult rat brain were immunoprecipitated with either anti-α5 or anti-IgG and immunoblotted for α5 and gephyrin. In both cortex and HPC, gephyrin coimmunoprecipitates with α5 (n = 3 animals). (C) Neurons were transfected with RFP-gephyrin and α2^WT, then fixed, stained under nonpermeabilized conditions with anti-GFP, then permeabilized and stained with anti-RFP. (D) Neurons were transfected with RFP-gephyrin, fixed, permeabilized, and stained for RFP and VGAT. (E) HEK cells were transfected with α5^WT, β3, and RFP-gephyrin. Lysates were then immunoprecipitated for GFP and immunoblotted for RFP and GFP. GFP immunoprecipitation resulted in coimmunoprecipitation of RFP-gephyrin. HEK cells transfected with α2^WT, β3, and RFP-gephyrin were used as a positive control.