Phosphorylation of p47phox in neutrophils from p67phox-deficient patients and from healthy donors. (A) Neutrophils from healthy donors (PMN-Control) and p67phox-deficient patients (PMN-CGD(p67−/−)) (15 × 106/400 µL) were incubated with fMLF (1 µM) or PMA (200 ng/mL) for 10 seconds and 10 minutes, respectively, 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 anti-gp91phox, anti-p67phox, anti-p47phox, anti-p40phox, and anti-p22phox antibodies. (B) Neutrophils from healthy donors (PMN-Control) and p67phox-deficient patients (PMN-CGD(p67−/−)) (15 × 106/400 µL) were incubated with fMLF (1 µM) or PMA (200 ng/mL) for 10 seconds and 10 minutes, respectively, 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. Experiments were repeated twice in duplicates.
