IL-4 regulates phagosomal cysteine cathepsin activity in a NOX2-dependent and -independent fashion. Total proteolytic efficiencies (A-B), cysteine cathepsin activities (C-D), and aspartic cathepsin activities (E-F) within phagosomes of untreated or IL-4–activated BMMØs were assessed in the presence of absence of functional NOX2. This was achieved by measuring the hydrolysis of particle-restricted fluorogenic substrates in WT and the NOX2-deficient Cybb^−/− BMMØs with or without inhibition of NOX2 by DPI (0.5μM). (A-B) General phagosomal proteolysis was assessed by measurement of fluorescence liberated through hydrolysis of particle-associated DQ-albumin. Relative activities of cysteine cathepsin (C-D) and aspartic cathepsins (E-F) were evaluated with the use of cathepsin B/L- and D/E-specific fluorogenic peptides bound to IgG-coupled experimental particles. (A,C,E) Real-time representative traces. RFU values are proportional to the degree of substrate hydrolysis. (B,D,F) Averaged activities relative to untreated WT controls are from 4 independent experiments. Activities 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. Error bars denote SEM. P values were determined by 1-way ANOVA.