Phosphorylation of p40^phox on T154, but not S315, is essential for NADPH oxidase activity in mouse neutrophils. (A) Lysates from p40^phox−/− BMNs reconstituted with GFP (empty vector [ev]), wt p40^phox (wt), p40^phox-T154A (T154A), or p40^phox-S315A (S315A) were subjected to SDS-PAGE and immunoblotted for phox components as described in “Methods.” A representative blot of 3 independent experiments is shown. Levels of p40^phox and p67^phox were normalized against levels of p47^phox, which was used as a loading control. Histograms represent relative levels of p40^phox (left) or p67^phox (right) as a percentage of wt. Data are mean ± SEM (n = 3 independent experiments performed in duplicate). (B-E) p40^phox−/− BMNs expressing GFP (empty vector [ev]; ○), wt p40^phox (▾), p40^phox-T154A (), or p40^phox-S315A (■) were pre-incubated with luminol/HRP before addition to fMLP (B) or PMA (E) or with luminol before addition to S aureus (C) or IgG-SRBCs (D), as described in “Methods.” ROS responses were measured by chemiluminescence as described in “Methods.” Shown are profiles of kinetics of ROS production, measured in RLU/s (mean ± range), from one representative experiment performed in duplicate, as well as total integrated ROS responses for 3-6 independent experiments (mean ± SEM), expressed as a percentage of the response in p40^phox−/− BMNs expressing wt p40^phox. The shaded area highlights the p40^phox-independent response. Where indicated, differences between means of wt and mutated versions of p40^phox are statistically significant. **P = .006; ***P = .000; as determined by a paired Student t test.