Figure 4. Co-immunoprecipitation of ASIC1a and GABA_A proteins in transfected HEK293 cells and DRG neurons.

A. Co-immunoprecipitation of ASIC1a and GABA_A proteins in transfected HEK293 cells. GABA_A specifically co-precipitates with ASIC1a in cells co-transfected with ASIC1a and GABA_A (both). n = 3. B1. DRG neurons were double-labeled with anti-ASIC1a and -GABA_AR antibodies. ASIC1a (red in left panel) as well as GABA_AR (green in middle panel) localized to the apical membrane of DRG neurons. The merge (right) of ASIC1a and GABA_AR images indicates that ASIC1a co-segregates with GABA_AR in the apical membrane in neurons (yellow). Nuclei were identified by DAPI staining (blue). Scale bars equal 50 µm. B2. GABA_A precipitates with ASIC 1a in primary cultured DRG neurons. DRG neurons lysates were immunoprecipitated (IP) with anti-GABA_A β_2/3 subunits polyclonal antibody in 8% SDS-PAGE gel, and the blot was then probed with anti- ASIC 1a polyclonal antibody (IB). In turn, ASIC 1a co-immunoprecipitates with GABA_A. Cell lysates were immunoprecipitated with ASIC 1a antibody in 6% SDS-PAGE gel, and the blot was probed with GABA_A β_2/3 antibody. These experiments were repeated three times with identical results.