FIGURE 4.
The p22phox mutant T147A is not phosphorylated by PKC-α or PKC-δ in vitro. Membrane protein from CHO 91/47/67 cells expressing no p22phox (Mock), wild type (wtp22), P156Q, T132A, or T147A p22phox was obtained as described under “Experimental Procedures.” p22phox was isolated by immunoprecipitation with monoclonal antibody 44.1 and phosphorylated by 100 ng of either PKC-α (A) or -δ (B). Reactions were stopped by the addition of Laemmli sample buffer and boiled for 10 min. Samples were subjected to SDS-PAGE and transferred to nitrocellulose, and radiolabeled p22phox was visualized by autoradiography. Blots were then subjected to Western blot analysis and probed for total p22phox. Blots are representative of at least two separate transfections.