Kinetics and concentration-dependent effect of PMA on p47phox phosphorylation and ROS production in human neutrophils. (A) Human neutrophils (15 × 106/400 µL) were incubated with PMA (200 ng/mL) for different times and the reaction was stopped by denaturation with 100 µL hot Laemmli sample buffer (5×). Homogenates were subjected to SDS-PAGE and western blotting using different anti-phospho-p47phox antibodies: anti-phospho-Ser304, anti-phospho-Ser315, anti-phospho-Ser320, anti-phospho-Ser328, and anti-phospho-Ser-345 antibodies and anti-p47phox antibody. (B) Neutrophils (0.5 × 106/500 µL) were incubated in HBSS for 5 minutes in the presence of 10 µM luminol at 37°C then stimulated with PMA (100 ng/mL); ROS production was measured by luminol-amplified chemiluminescence over time. (C) Human neutrophils (15 × 106/400 µL) were incubated with different concentrations of PMA for 10 minutes and the reaction was stopped by denaturation with 100 µL hot Laemmli sample buffer (5×). Homogenates were subjected to SDS-PAGE and western blotting using the same antibodies as in panel A. (D) ROS production of neutrophils (0.5 × 106/500 µL in HBSS) was measured as in panel B. Experiments were repeated 3 times (n = 3, mean ± SEM).
