Figure 7
Figure 7. IL-4 activation enhances the reductive capacity of the phagosome in a NOX2-dependent fashion. The reductive capacity of the phagosome was assessed by measuring intraphagosomal rates of disulfide reduction in untreated and IL-4–activated, WT, and Cybb−/− BMMØs. (A-B) Measurement of fluorescence liberated through intraphagosomal reduction of a modified fluorogenic cystine-based reagent covalently bound to IgG-coupled experimental particles after phagocytosis, relative to calibration fluorescence. Inhibition of NOX2 was achieved with DPI (0.5μM) where indicated. (A) Real-time representative traces. RFU values are proportional to the degree of substrate reduction. (B) Averaged rates relative to DMSO-treated untreated WT samples from 4 independent experiments, error bars denote SEM, P values were determined by ANOVA. (C-D) Assessment of intraphagosomal efficiency to reduce inter-molecular disulfide bonds of particle-restricted IgG. Biotinylated anti-BSA IgG was used to opsonize BSA-coupled experimental particles and given to BMMØs in the presence of protease inhibitors. After 1 hour at 37°C, cells were lysed in nonreducing sample buffer and protein separated by SDS-PAGE. Biotinylated whole and heavy-chain IgG were identified by Western blot with the use of streptavidin-HRP and standard chemiluminescent detection. Despite the presence of protease inhibitors, increased loss of IgG in both whole and heavy chain forms was noted in IL-4–treated Cybb−/− BMMØs. Hence, the efficiency of IgG dissociation was assessed with the use of heavy chain density normalized to whole IgG band density. (C) Representative Western blot of reduction of whole IgG molecule in untreated and IL-4–activated BMMØs derived from WT and Cybb−/− mice. (D) Efficiency of reduction of IgG as expressed by ratio of heavy chain band density to whole IgG molecule band density. Band density was determined by calculation of pixel volume by the use of Quantity One analysis software (Bio-Rad). Graphs represent averaged data from 6 independent experiments. Error bars represent SEM. P values were determined by paired t test.

IL-4 activation enhances the reductive capacity of the phagosome in a NOX2-dependent fashion. The reductive capacity of the phagosome was assessed by measuring intraphagosomal rates of disulfide reduction in untreated and IL-4–activated, WT, and Cybb−/− BMMØs. (A-B) Measurement of fluorescence liberated through intraphagosomal reduction of a modified fluorogenic cystine-based reagent covalently bound to IgG-coupled experimental particles after phagocytosis, relative to calibration fluorescence. Inhibition of NOX2 was achieved with DPI (0.5μM) where indicated. (A) Real-time representative traces. RFU values are proportional to the degree of substrate reduction. (B) Averaged rates relative to DMSO-treated untreated WT samples from 4 independent experiments, error bars denote SEM, P values were determined by ANOVA. (C-D) Assessment of intraphagosomal efficiency to reduce inter-molecular disulfide bonds of particle-restricted IgG. Biotinylated anti-BSA IgG was used to opsonize BSA-coupled experimental particles and given to BMMØs in the presence of protease inhibitors. After 1 hour at 37°C, cells were lysed in nonreducing sample buffer and protein separated by SDS-PAGE. Biotinylated whole and heavy-chain IgG were identified by Western blot with the use of streptavidin-HRP and standard chemiluminescent detection. Despite the presence of protease inhibitors, increased loss of IgG in both whole and heavy chain forms was noted in IL-4–treated Cybb−/− BMMØs. Hence, the efficiency of IgG dissociation was assessed with the use of heavy chain density normalized to whole IgG band density. (C) Representative Western blot of reduction of whole IgG molecule in untreated and IL-4–activated BMMØs derived from WT and Cybb−/− mice. (D) Efficiency of reduction of IgG as expressed by ratio of heavy chain band density to whole IgG molecule band density. Band density was determined by calculation of pixel volume by the use of Quantity One analysis software (Bio-Rad). Graphs represent averaged data from 6 independent experiments. Error bars represent SEM. P values were determined by paired t test.

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