Figure 2. Use of an antibody to phospho-Ser345 demonstrates that GM-CSF and TNF-α induce phosphorylation of p47^phox on Ser345 in a concentration- and time-dependent manner.

(A) Neutrophils (1 × 10⁷ cells/ml) were incubated with various concentrations of GM-CSF for 20 minutes or were incubated with GM-CSF (12.5 ng/ml) for the times indicated. Cells were lysed, and proteins from 4 × 10⁵ cells were analyzed with SDS-PAGE and immunoblotting with anti–phospho-Ser345 antibody (p-Ser345) or anti-p47^phox antibody (p47^phox). (B) Neutrophils (1 × 10⁷ cells/ml) were incubated with various concentrations of TNF-α for 20 minutes or were incubated with TNF-α (10 ng/ml) for the times indicated. Cells were lysed, and proteins from 4 × 10⁵ cells were analyzed with SDS-PAGE and immunoblotting with anti–phospho-Ser345 antibody or anti-p47^phox antibody. Western blots from different experiments were scanned; phosphorylated and total p47^phox were quantified by densitometry; and the intensity of phosphorylated p47^phox was corrected for the amount of p47^phox. Results are expressed as mean ± SEM (n = 3). *P < 0.05 compared with untreated neutrophils or time 0 minutes.