Mutation or deletion of the SH3 domain of p40^phox does not impair ROS production in mouse neutrophils. (A) p40^phox−/− BMNs reconstituted with GFP (empty vector [ev]), wt p40^phox (wt), p40^phox-ΔSH3 (ΔSH3), or p40^phox-W207Y (W207Y) were sonicated, subjected to SDS-PAGE, and immunoblotted for phox components as described in “Methods.” A representative blot of 2 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 ± range (n = 2 independent experiments performed in duplicate). (B-E) p40^phox−/− BMNs expressing GFP (ev; ○), wt p40^phox (▾), p40^phox-ΔSH3 (), or p40^phox-W207Y (■) were used in assays for ROS production as described in Figure 3. Shown are through 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 a minimum of 3 independent experiments (mean ± SEM; ev: n = 8-9; wt: n = 9; ΔSH3: n = 9; W207Y: n = 3), 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 = .009; as determined by a repeated measures ANOVA.