Analysis of the expression of NADPH oxidase in G6pc3^−/− neutrophils. Annexin V–depleted BM neutrophils were isolated from 6- to 8-week-old unaffected (+/+) and G6pc3^−/− (−/−) littermates as described in “Depletion of apoptotic cells from neutrophils.” (A) Quantification of gp91^phox, p22^phox, and p47^phox mRNA by real-time RT-PCR. Data represent the mean ± SEM of 3 independent experiments. *P < .05, **P < .005. (B) Representative immunofluorescence analysis of gp91^phox (red fluorescence) or p22^phox (green fluorescence) and DAPI nuclei staining (blue fluorescence) at 400× magnification. (C) Western blot analysis of protein extracts using antibodies against gp91^phox, p22^phox, p47^phox or β-actin. Data from 2 pairs of littermates are shown and each lane contains 50 μg of protein. The relative protein levels of gp91^phox, p22^phox, p47^phox were quantified by densitometry of 4 separate pairs of Western blots. *P < .05; **P < .005. (D) Representative immunofluorescence analysis of p47^phox (green fluorescence), pan Cadherin membrane staining (red fluorescence), and DAPI nuclei staining (blue fluorescence) at 400× magnification.