Effects of R58A mutation in p40^phox on mouse neutrophil ROS responses. (A) Bone marrow neutrophils derived from wild-type (p40^phox+/+), p40^{phoxR58A/R58A}, or p40^phox−/− animals were sonicated into SDS sample buffer, subjected to SDS-PAGE, and immunoblotted for p40^phox, p47^phox, and p67^phox as described in “Western blotting.” (Bi,ii and Ci). Primed bone marrow neutrophils from p40^phox+/+ (♦) and p40^{phoxR58A/R58A} () mice were prepared and preincubated with luminol in the absence (solid symbols) or presence (open symbols) of 100 nM wortmannin, as described in “Preparation of cells” and “Measurement of ROS production.” Cells (5 × 10⁵/well) were added to 10⁷ serum-opsonized S aureus (Bi) or E coli (Bii), and light emission measured over 40 minutes as described in Figure 1. (Bi,ii) Open circles represent ROS production in the absence of bacteria for p40^phox+/+ (black) and p40^{phoxR58A/R58A} (gray) neutrophils. Rate measurements of ROS production (mean ± range) from a single experiment representative of 3. (Ci) Accumulated light emissions over the 40-minute measurement period combined from 3 individual experiments and expressed as a percentage of WT responses (mean ± SEM). (Cii) Primed bone marrow neutrophils from p40^phox+/+ and p40^{phoxR58A/R58A} mice were incubated, in the presence of luminol and HRP (18.75 U/mL), with fMLP (10 μM final) and ROS production measured as described in Figure 1, except light emission was recorded for 3 minutes. Data presented are accumulated light emissions from 3 independent experiments (mean ± SEM).