Stim1−/−neutrophils have defective activation of PKCα/β and fail to phosphorylate the p40phoxsubunit of the NADPH oxidase. (A) WT and stim1−/− neutrophils were plated onto milk- or pRGD-coated coverslips for 10 minutes, fixed, and then stained with anti-PKCα or anti-PKCβ antibodies. (B) Total cellular membranes were isolated from WT or stim1−/− neutrophils, either resting (control) or stimulated for 3 minutes with fMLF. Equal amounts of protein were electrophoresed and then immunoblotted with anti-PKCα, anti-PKCβ, or antiactin to verify equal loading. Immunoblots were imaged with a Licor system. (C) WT and PKCα/β−/− neutrophils were plated on fibrinogen in the presence or absence of TNF-α. Reduction of cytochrome C was determined in a Spectramax plate reader at 37°C. Statistical analysis at peak response was P < .001 between WT and PKCα/β−/− cells. (D) Upper panel: WT neutrophils in HBSS with or without Ca2+ were plated on 0.2% milk–coated plates with fMLF or on fibrinogen-coated plates with TNF-α or thapsigargin for 15 minutes. Total protein was immunoblotted with the phospho-specific anti-p40phox monoclonal antibody. Lower panels: WT or stim1−/− neutrophils were stimulated as indicated, and phosphorylation of the p40phox and p47phox proteins was determined. The same blot was reprobed with anti-ERK antibodies to verify equal loading. Immunoblots were imaged by electrochemiluminescence detection. Lanes shown for p47phox are from the same experiment but run on different gels. Data shown are representative of 2 to 4 independent experiments.