Association of Pak with p22^phox. (A) Cos cells expressing functional flavocytochrome b were transfected with vector (lane 1), wild-type Myc-Pak1 (lane 2), constitutively active Myc-Pak1 F107 E423 (lane 3), dominant-negative Myc-Pak1 A299 (lane 4), Myc-Pak1 (aa 206-545) (lane 5), or Myc-Pak1 (aa 1-205) (lane 6). Anti-p22^phox immunoprecipitates were prepared after 24 hours and probed with anti-Myc antibody for Pak detection (top panel). The middle and the bottom panels show Myc-Pak1 protein expression levels and p22^phox protein in the IP, respectively. Representative of 3 independent experiments and verified twice in Cos cells stably expressing p22^phox alone. Immunoblots of cell lysates are indicated by IB. (B) GST beads (lanes 1-2) or GST-Pak1 beads (lanes 3-4) were incubated with buffer (lanes 1,3) or purified flavocytochrome b (lanes 2,4). After washing, proteins bound to the beads were eluted and immunoblotted with anti-gp91^phox or anti-p22^phox antibodies. Control blots for the flavocytochrome b are shown in lane 5. Equal amounts of recombinant GST or GST-Pak1 bound to beads were verified by Coomassie blue staining (bottom panels). A representative data set of 4 independent experiments is shown.