IL-4 decreases the NOX2-dependent phagosomal respiratory burst and the expression of the NOX2 subunit gp91^phox. (A-B) After phagocytosis of experimental particles, the phagosomal respiratory burst was assessed by measurement of fluorescence liberated by oxidation of particle-associated H2HFF-OxyBURST substrate in WT and the NOX2-deficient Cybb^−/− BMMØs. Inhibition of NOX2 was achieved with DPI (0.5μM) where indicated. (A) Real-time traces of phagosomal respiratory burst. RFU values are proportional to the degree of substrate oxidation. (B) Averaged rates of substrate oxidation from 4 independent experiments. Rates of oxidation were determined by calculation of the slope of the linear portion of the real-time trace (as described by y = mx + c, where y = relative fluorescence, m = slope, and x = time) and expressed relative to untreated WT controls. (C) Assessment of extracellular ROS released by BMMØs in response to PMA (1μM) was achieved by measurement of luminol-generated luminescence over 10 minutes and expressed as averaged counts per second from 4 independent experiments. Error bars denote SEM. P values were determined by 1-way ANOVA. (D) Total mRNA levels of major NOX2 subunits were determined by quantitative PCR. Averaged relative mRNA expression level from 5 independent QPCR experiments is shown. Relative expression was expressed as mRNA levels relative to 18S and presented relative to untreated BMMØs. Error bars denote SEM. P values were determined by 1-way ANOVA. (E-F) Total cellular abundance of major NOX2 subunits was determined by semiquantitative Western blotting of whole cell lysates. (E) Representative Western blot images. (F) Average of band relative density from 3 independent Western blot experiments. (G-H) Phagosomal recruitment of major NOX2 subunits was determined by semiquantitative Western blotting of phagosomes isolated 30 minutes after particle uptake. (G) Representative Western blot images. (H) Average of band relative density from 4 independent Western blot experiments. Relative density was determined by calculation of pixel volume for treated sample relative to untreated BMMØ sample by the use of Quantity One analysis software (Bio-Rad). Error bars denote SEM, P values were determined by paired t test. (I) Relative recruitment of p67^phox and p47^phox (from Figure 3H) normalized to the abundance of phagosomal gp91^phox in IL-4–activated BMMØs.