Pin1 facilitates phosphorylation of p47phox by PKC, both in vitro and in intact neutrophils. (A) p47phox was phosphorylated with p38MAPK and then incubated with or without Pin1 preincubated with or without juglone. Where indicated, PKC was added in the presence of 32P-γ-ATP. Proteins were analyzed by SDS-PAGE, autoradiography, and immunoblotting (IB). (B) Phosphorylated p47phox peptides containing phospho-Ser315 (p-Ser315), p-Ser320, p-Ser328, or p-Ser345 were subjected to 16% SDS-PAGE, transferred to polyvinylidene difluoride membranes, and revealed with anti p-Ser315, p-Ser320, p-Ser328, or p-Ser345 antibodies. (C) Neutrophils were incubated with or without GF109203X (GFX) (5μM) for 15 minutes, stimulated with PMA, and proteins were analyzed with SDS-PAGE and Western blotting using anti–phospho-Ser315, anti–phospho-Ser320, anti–phospho-Ser328, and anti-p47phox antibodies. (D) Neutrophils were treated with TNF-α and fMLF, alone or together, in the absence or presence of juglone. Neutrophils were then lyzed, and proteins were analyzed with SDS-PAGE and Western blotting with anti–phospho-Ser315, anti–phospho-Ser320, anti–phospho-Ser328, anti–phospho-Ser345, and anti-p47phox antibodies. All experiments are representative of 3.
