Analysis of NADPH and the expression of NADPH oxidase in G6PT-deficient neutrophils of GSD-Ib patients. Annexin V–depleted peripheral blood neutrophils isolated from HDs and GSD-Ib patients were used in the study. (A) Levels of neutrophil NADPH. Data represent the mean ± SEM for HDs (n = 7) and GSD-Ib patients (n = 6). (B) Quantification of gp91^phox, p22^phox, and p47^phox mRNA by real-time RT-PCR. Data represent the mean ± SEM for HDs (n = 10) and GSD-Ib patients (n = 12). (C) Western blot analysis of protein extracts using antibodies against gp91^phox, p22^phox, p47^phox, or β-actin. Each lane contains 50 μg of protein. (D) The relative protein levels of gp91^phox, p22^phox, and p47^phox were quantified by densitometry. Data represent the mean ± SEM for HDs (n = 6) and GSD-Ib patients (n = 6). (E) Confocal analysis of p47^phox (green fluorescence), pan Cadherin membrane staining (red fluorescence), and DAPI nuclei staining (blue fluorescence) at original magnification ×630 and quantification of the relative integrated fluorescence intensity by ImageJ. The p47^phox translocation from the cytoplasm to the plasma membrane was demonstrated by colocalization of p47^phox with the plasma membrane marker pan Cadherin. **P < .005.