Pak/p22^phox binding is independent of p47^phox association with flavocytochrome b₅₅₈. (A) Cos cells expressing flavocytochrome b were cotransfected with vector (lane 1), Myc-Pak1 F107, E423 (lane 2), or Myc-Pak1 (aa 206-545) (lane 3), and wild-type p47^phox (lanes 1-3). After 30 hours, the cells were treated with vehicle (dimethyl sulfoxide [DMSO]) or PMA (1 ng/mL) for 10 minutes before lysis. The top panel shows Myc-Pak1 in p22^phox immunoprecipitates (IP) by probing with anti-Myc antibody. Myc-Pak1 and p47^phox expression levels are shown in the middle and bottom panel (immunoblot IB). (B) Anti-p22^phox immunoprecipitates were blotted and probed with anti-p47^phox antibody after PMA stimulation or vehicle (DMSO) treatment. A representative data set of 3 independent experiments is shown.