Fig. 3.
PSD95 in the varicosity membrane associates with both serine847-phospho-nNOSα dimer and dephospho-nNOSα dimer. Purified varicosity membrane samples were immunoprecipitated with anti-PSD95 antibody. The immunoprecipitate was immunoblotted with anti-nNOS1–20 antibody. 320-kDa band of nNOSα dimer was seen, suggesting that PSD95 is associated with nNOSα dimer in the varicosity membrane. Incubation with IgG served as negative control. Furthermore, varicosity membrane samples immunoprecipitated with anti-PSD95 was again immunoprecipitated with anti-serine847-phospho-nNOS antibody. The resulting immunoprecipitate and supernatant were separately loaded for SDS-PAGE and probed with anti-nNOS1–20 antibody. Both the immunoprecipitate and the supernatant showed 320-kDa bands, suggesting that the PSD95 in varicosity membrane is associated with both serine847-phospho-nNOSα dimer and dephospho-nNOSα dimer. When probed with anti-calmodulin (CaM) antibody, in the presence of Ca2+, only the supernatant showed a 17-kDa band. IgG negative control and purified bovine brain CaM as a positive control for the 17-kDa signal were used for the coimmunoprecipitation experiments.