Phosphorylation of recombinant p47phox by purified PKC isoforms in vitro and effect of p67phox and p40phox. Recombinant p47phox protein was incubated without or with p67phox or p40phox or p67phox+p40phox for 10 minutes; PKCα, PKCβ, PKCδ, or PKCζ were added for 5 minutes and the reaction was started by adding ATP, followed by incubation at 30°C for 15 minutes. The reactions were terminated by addition of Laemmli 5× sample buffer and denaturation at 100°C. Proteins were subjected to SDS-PAGE and western blotting using different anti-phospho-p47phox antibodies: anti-phospho-Ser304, anti-phospho-Ser315, anti-phospho-Ser320, anti-phospho-Ser328 antibodies and anti-p47phox antibody. Experiments were repeated 3 times.