Stim1^−/−neutrophils have defective activation of PKCα/β and fail to phosphorylate the p40^phoxsubunit 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-p40^phox monoclonal antibody. Lower panels: WT or stim1^−/− neutrophils were stimulated as indicated, and phosphorylation of the p40^phox and p47^phox 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 p47^phox are from the same experiment but run on different gels. Data shown are representative of 2 to 4 independent experiments.