Pin1 interacts with p47phox via phosphorylated Ser345. (A) Neutrophils were treated with TNF-α alone, fMLF alone, or TNF-α followed by fMLF (TNF-α + fMLF), and then lysed and incubated with GST-Pin1 in the presence of glutathione beads. The beads were then washed, Pin1 was released by thrombin cleavage, and proteins were analyzed by SDS-PAGE and Western blot (IB). (B) Recombinant p47phox was phosphorylated with p38MAPK and then repurified and incubated with GST-Pin1 and glutathione-agarose beads. The beads were washed 3 times, and proteins were analyzed by SDS-PAGE and immunoblotting (IB). (C) p47phox peptides containing phosphorylated or nonphosphorylated Ser345 were coupled to ovalbumin, spotted on nitrocellulose membranes, and overlaid with recombinant Pin1. Pin1 was detected with a specific antibody. Experiments are representative of 3.