Effect of p47^phox on serine residues 303, 304, and 379 NADPH oxidase activation in COS-phox cells.

COS cells stably expressing gp91^phox and p22^phox were transiently transfected with wild-type p67^phox plus either wild-type p47^phox (p47 wt) or p47^phoxmutants Ser303Ala/Ser304Ala, Ser303Glu/Ser304Glu, or Ser379Ala, or empty pRK5 vector. Cells were analyzed 24 hours after transfection. Following stimulation with 0.4 μg/mL PMA, superoxide production was determined by reduction of ferricytochrome c. Measured rates of superoxide production were normalized to that of cells expressing wild-type p47^phox, and data were also normalized on the basis of the level of expression of the mutants compared with wild-type p47^phox expression. Relative protein expression levels compared with wild-type p47^phox were 76 ± 22% for p47^phox Ser303Ala/Ser304Ala, 82 ± 27% for p47^phox Ser303Glu/Ser304Glu, and 90 ± 29% for p47^phox Ser379Ala, as determined by densitometry performed on blots of whole-cell lysates following SDS–PAGE (not shown). Data represent mean ± SD; n = 3. *Superoxide production is significantly lower for cells transfected with p47^phox Ser303Ala/Ser304Ala, p47^phox Ser379Ala, or empty vector, compared with cells expressing wild-type p47^phox or p47^phox Ser303Glu/Ser304Glu (P < .01, Student-Newman-Keuls multiple-comparisons test). Superoxide production by the cells transfected with wild-type p47^phox was 1.8 ± 0.3 nmol/10⁷cells per minute.