Direct interaction between eNOS and porin. (A) Western blot analysis of eNOS immunoprecipitates. In each lane, 500 μg Brij 96 lysates from human endothelial cells were immunoprecipitated with mouse preimmune IgG or anti-eNOS polyclonal antibody. For the lysate lane, 30 μg Brij 96 lysates were used. Immunoprecipitated proteins were subjected to immunoblotting with anti-porin (Upper) and anti-eNOS (Lower) antibodies. (B) Western blot analysis of porin immunoprecipitates. Brij 96 lysates (500 μg) from human endothelial cells were immunoprecipitated with mouse preimmune IgG or anti-porin mAb, and 30 μg Brij 96 lysates were used in lysate lane. Immunoprecipitated proteins were subjected to immunoblotting with anti-eNOS (Upper) and anti-porin (Lower) antibodies. (C) Recombinant eNOS incubated with either GST or GST-porin, and proteins were precipitated with glutathione-Sepharose 4B beads. The precipitated proteins were separated through SDS/PAGE, and Western blot analysis was done with anti-eNOS monoclonal antibody. IP, immunoprecipitation; IB, immunoblotting.