Oxidative stress and intrinsic mitochondrial apoptotic pathway in G6pc3^−/− neutrophils. BM neutrophils were isolated from 6- to 8-week-old control (+/+) and G6pc3^−/− (−/−) littermates as described in “Neutrophil isolation.” (A) Quantitative flow cytometric analysis of neutrophil carboxy-DCF staining. Data are the mean ± SEM of 4 independent experiments. (B) Western blot analysis of protein extracts of neutrophils using antibodies against Mn-SOD or β-actin. Each lane contains 50 μg protein. The relative protein levels of Mn-SOD were quantified by densitometry of 3 separate pairs of Western blots. (C) Representative confocal microscopic analysis of Bax (green fluorescence), COX IV (red fluorescence, mitochondria), and DAPI nuclei (blue fluorescence) staining (original magnification ×1000). (D) Western blot analysis of protein extracts of neutrophils using antibodies against Bax, Smac/Diablo, Omi/HtrA2, or β-actin. Each lane contains 50 μg protein. The relative protein levels of Bax, Smac/Diablo, or Omi/HtrA2 were quantified by densitometry of 3 or 4 separate pairs of Western blots. (E) Quantitative flow cytometric analysis of cytochrome c release. Data are the mean ± SEM of 3 independent experiments. (F) Flow cytometry, immunoprecipitation, and Western blot analysis of immunoprecipitates using an antibody against active caspase-9 and a horseradish peroxidase-conjugated secondary antibody. Data for flow cytometric analysis represent the mean ± SEM of 4 independent experiments. (G) Quantitative flow cytometric analysis of active caspase-3. Data are the mean ± SEM of 4 independent experiments. **P < .005. *P < .05.