Mo-DC express high levels of active NOX5. Total RNA was extracted from freshly isolated Mos (MONO), immature Mo-DCs (GM-CSF/IL-4), and mature Mo-DCs (LPS) by standard techniques. mRNA levels for (A) NOX1-5 were determined by endpoint PCR using specific primers. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as loading control. The size of the products is represented in base pairs (bp). (B) Real-time PCR of NOX2 and NOX5. Relative expression to control (MONO) levels for each gene was calculated using the 2^−ΔΔCt method, with normalization to the average hypoxanthine phosphoribosyltransferase 1 (HPRT) and GAPDH housekeeping genes. One-way ANOVA with Dunn post-test, *P < .05. (C) Representative western blot analysis for NOX2 (gp91phox), NOX5, and actin (n = 3) in Mos and DC. (D) Time course of expression of NOX5 in Mos treated with GM-CSF/IL-4. (E) Representative western blot (n = 3) for NOX5 and actin in MONO, Mo-DC (GM-CSF/IL-4) cells, and macrophage (GM-CSF). (F) Representative western blots for NOX5 expression in freshly isolated pDC and mDC (n = 2). Quantification of these data can be found in supplemental Figure 3. (G-H) Mos were treated with GM-CSF/IL-4 for 48 hours, incubated in the absence or presence of 20 µM TAT-NOX2 peptide, stimulated with 1 µM ionomycin (G) or 1 µM fMLF (H), and ROS production was measured using luminol-amplified chemiluminescence (n = 3).