Figure 4.

Effect of PMA and GF109203X on gp91^phox/NOX2 phosphorylation and ROS production in human neutrophils. A) Kinetics of PMA-induced gp91^phox/NOX2 phosphorylation in human neutrophils. ³²Pi-labeled neutrophils (5×10⁷ cells/ml) were incubated with PMA (200 ng/ml) for various times. Gp91^phox/NOX2 was immunoprecipitated, and samples were subjected to SDS-PAGE, blotted on nitrocellulose, and detected by autoradiography (³²P-NOX2) and Western blotting (NOX2). B) Kinetics of PMA-induced NADPH oxidase activation in human neutrophils. Human neutrophils (5×10⁵ cells/0.5 ml Hank’s buffer) were treated with buffer or PMA (200 ng/ml), and luminol-amplified chemiluminescence was measured. C) Effect of the PKC inhibitor GF109203X on gp91^phox/NOX2 phosphorylation in human neutrophils. ³²Pi-labeled neutrophils were incubated in the presence or absence of GF109203X (1–8 μM) for 15 min, stimulated with PMA (200 ng/ml) for 8 min, and gp91^phox/NOX2 was immunoprecipitated from lysates. Lysates were analyzed by autoradiography (³²P-NOX2) and immunoblotting (NOX2). Data are representative of 3 experiments.