Concentration-dependent effect of p67phox on p47phox phosphorylation by PKC isoforms in vitro and effect of denatured p67phox. Recombinant p47phox protein was incubated without or with different concentrations of p67phox or with heat-denatured p67phox for 10 minutes; PKCα, PKCβ, PKCδ, or PKCζ was added, incubated 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 the addition of 5× Laemmli sample buffer and denaturation at 100°C. Proteins were subjected to SDS-PAGE and western blotting using various anti-phospho-p47phox antibodies. Results with anti-phospho-Ser328 antibody are shown. Experiments were repeated 3 times.