Analysis of 2-DG uptake, the expression of GLUT1, and intracellular G6P, lactate, ATP, and glycogen levels 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.” Freshly isolated annexin V–depleted neutrophils were used for 2-DG uptake, quantitative RT-PCR, and Western blot analyses. Immunofluorescence analysis of GLUT1 and measurement of G6P, lactate, ATP, and glycogen were conducted in annexin V–depleted neutrophils that were incubated for 30 minutes at 37°C in glucose-containing RPMI-1640 medium as described in “Immunofluorescene microscopic analyses”; “G6P, lactate, and glycogen determination”; and “Total ATP determination.” For 2-DG uptake and quantitative RT-PCR, the data represent the mean ± SEM of 3 independent experiments. **P < .005. For Western blot and immunofluorescence analysis, at least 3 separate experiments were conducted in which each mouse was assessed individually. (A) Uptake of 2-DG. (B) Quantification of GLUT1 mRNA by real-time RT-PCR. (C) Western blot analysis of protein extracts of annexin V–depleted BM neutrophils using antibodies against GLUT1 or β-actin. Each lane contains 50 μg of protein. The relative GlUT1 protein levels were quantified by densitometry of 4 separate pairs of Western blots. *P < .05. (D) Representative immunofluorescence of GLUT1 staining (green fluorescence), pan Cadherin membrane staining (red fluorescence), and DAPI nuclei staining (blue fluorescence) at 400× magnification. (E) G6P, lactate, ATP, and glycogen levels. Data represent the mean ± SEM of 4 independent experiments. *P < .05.