Kinetics and concentration-dependent effect of fMLF on p47phox phosphorylation and ROS production in human neutrophils. (A) Human neutrophils (15 × 10⁶/400 µL) were incubated with fMLF (1 µM) 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 and stimulated with fMLF (1 µM); ROS production was measured by luminol-amplified chemiluminescence over time. (C) Human neutrophils (15 × 10⁶/400 µL) were incubated with different concentrations of fMLF for 10 seconds 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 HBSS) was measured as in panel B. Experiments were repeated 3 times (n = 3, mean ± standard error of the mean [SEM]).