Figure 7.
Co-assembly of α3-containing GABAA receptors with GABARAP can be disrupted by short-chain interfering peptides. A, Western blots of lysates from HEK 293 cells transfected with HA-GABAR-α3, GABAR-β2, and GABAR-γ2 (short) to form α3β2γ2 GABAR channels that have been coexpressed with either Gephyrin-YFP (left) or GABARAP-CFP (right). A, Left, Blot with eluates and inputs (n = 3) of cell lysates immunoprecipitated with an anti-HA antibody and analyzed by immunoblotting with an anti-GFP antibody. YFP is presented as a negative control (note that the anti-GFP antibody recognizes both YFP and CFP). Right, Blot with eluates and inputs (n = 3) of cell lysates immunoprecipitated with an anti-HA antibody and analyzed by immunoblotting with an anti-GFP antibody. B, Primary amino-acid sequence of the K1-GABARAP blocking peptide and the α3-derived-gephyrin blocking peptide. C, Scrambled GABARAP peptide or GABARAP peptide were added to lysates from the same transfections and pulled down with anti-HA antibody. Immunoblotting was performed with an anti-GFP antibody, as in A. D, Bar graph comparing GABARAP immunoblot levels after pre-incubation with GABARAP or scrambled peptide. p < 0.001, n = 4, Student's t test. Error bars indicate SEM.