p67phox knockdown reduces the phosphorylation of p47phox and its expression increases this process in COSphox cell lines. (A) COSphox cells (expressing gp91phox, p22phox, p47phox, and p67phox) were transiently transfected with siRNA p67phox or scrambled siRNA as control. Forty-eight hours after transfection, cell homogenates were prepared and subjected to western blotting using anti-p47phox and anti-p67phox antibodies to verify the effect of siRNA-mediated knockdown (A; bottom) and with different anti-phospho-p47phox antibodies as indicated (A; top). Images shown are representative western blots from 3 independent experiments. (B) The NIH Image J software was used for densitometry analysis and the results expressed as mean ± SEM are shown (n = 3; *P < .05). (C) COS-7 cells were either transiently transfected with gp91phox cDNA in pEF-PGKpac and p22phox/p47phox/p67phox cDNA in pRK5 or transiently transfected with only gp91phox cDNA in pEF-PGKpac and p22phox/p47phox cDNA in pRK5. Twenty-four hours after transfection, the cells were stimulated with vehicle or PMA (200 ng/mL final) for 30 minutes. Cell homogenates were prepared and subjected to western blotting using different anti-phospho-p47phox antibodies, as indicated. (D) Images shown are representative western blots from 3 independent experiments. The NIH Image J software was used for densitometry analysis (n = 3; *P < .05).