G6pc3^−/− BM neutrophils display enhanced ER stress and apoptosis. Bone marrow (BM) neutrophils were isolated from 6- to 8-week-old unaffected (+/+) and G6pc3^−/− (−/−) littermates as described in “Isolation of murine bone marrow and human blood neutrophils.” (A) Hema 3–stained cytospins of BM neutrophils. (B) Western blot analysis of protein extracts of neutrophils using antibodies against Gr-1, gelatinase, or β-actin. Data from 2 pairs of littermates are shown and each lane contains 80 μg of protein. (C) A plot of BM neutrophil counts in control and G6pc3^−/− mice determined by flow cytometry analysis using anti–Gr-1 and anti-CD11b antibodies (n = 4). (D) Western blot analysis of protein extracts of neutrophils using antibodies against GRP78, GRP170, PDI, or β-actin. Data from 2 pairs of littermates are shown and each lane contains 50 μg of protein. (E) Quantification of apoptotic cells (annexin V⁺) in BM neutrophils of control and G6pc3^−/− mice determined by flow cytometric analysis. At least 5000 cells were used for each determination (n = 4). (F) The DEVD-cleaving activity of active caspase-3 in protein extracts of BM neutrophils. Data represent the mean ± SEM of 3 independent experiments. *P < .05, **P < .005.