GluR2/GAPDH interaction occurs extracellularly. A, Rat hippocampal neurons were incubated with sulfo-NHS-LC biotin to label cell surface proteins. GAPDH that co-immunoprecipitated with GluR2 antibody was examined in both non-biotinylated (NB) and biotinylated (B) proteins. B, Using a rabbit anti-GAPDH antibody, GAPDH was immunoprecipitated from the conditioned medium (CM; medium incubated with neurons/cells for 24 hours) of primary cultures of rat hippocampus but not from fresh medium. A mouse GAPDH antibody was used for Western blotting and rabbit IgG was used as negative control. C, Western blot analysis of GAPDH and α-tubulin in concentrated conditioned medium of non-transfected HEK-293 T cells (non-T) and HEK-293 T cells transfected with GluR1/2 subunits (AMPAR), in the presence or absence of glutamate (AMPAR + Glut). Cell lysates were used as controls. D-E: Coimmunoprecipitation of GAPDH by primary antibody against GluR2 subunit (with or without glutamate treatment) from HEK-293 T cells expressing GluR1/2 subunits (D) and hippocampal neurons (E) pre-treated with GluR2NT1-3–2 or GluR2NT1-3–2-scram peptides (top panels). Each Coimmunoprecipitation was in parallel with Western blot analysis of the directly immunoprecipitated proteins (bottom panels). All western blot analysis and co-immunoprecipitation assays in this figure are representative of at least 3 independent experiments.