FIG. 3.

Nox2 facilitates superoxide production in the endosomal compartment following IL-1β stimulation. (A) Western blot analysis of p47phox, p67phox, Rac1, MyD88, TRAF6, Rab5, and Cu/ZnSOD proteins in the isolated vesicular fraction of MCF-7 cells infected with Ad.BglII (control virus) or Ad.Dyn(DN) or treated with SOD and/or catalase proteins prior to IL-1β treatment for 20 min. (B) Time course of IL-1R1, Nox2, MyD88, and TRAF6 recruitment into the vesicular compartment following IL-1β treatment in the presence or absence of purified bovine Cu/ZnSOD and catalase proteins added to the medium. Isolated vesicular fractions were analyzed by Western blotting for the indicated proteins at various times post-IL-1β stimulation. (C and D) MCF-7 cells were transfected with a Nox2 expression plasmid (pNox2) or an irrelevant plasmid (pcDNA). At 48 h posttransfection, cells were stimulated with IL-1β and vesicular fractions were isolated at 20 min poststimulation. (C) NADPH-dependent ·O₂⁻ production by Iodixanol gradient fractions following transfection with the indicated constructs. (D) Western blotting evaluating Nox2 expression in whole-cell lysates and isolated peak vesicular fractions 2 to 4 (longer exposures of lanes 6 and 9 detect endogenous levels of Nox2). Fully processed Nox2 migrates at ∼95 kDa. (E and F) MCF-7 cells were transfected with Nox2 siRNA or a scrambled (Scr) control siRNA 48 h prior to IL-1β treatment. (E) NADPH-dependent ·O₂⁻ production of Iodixanol gradient fractions following transfection with the indicated siRNA constructs. Inset depicts Western blot expression of total cellular endogenous Nox2 protein from cells transfected with Nox2 siRNA or scrambled siRNA. (F) Western blotting for Nox2, evaluating the effect of Nox2 or scrambled siRNA transfection on the recruitment of endogenous Nox2 (lanes 1 to 4), or transfected Nox2 (from a pNox2 expression plasmid) (lanes 6 to 9), into the peak Iodixanol vesicular fractions (2 to 4). (G) MCF-7 cells were transfected with Nox2 or scrambled siRNAs 48 h prior to IL-1β treatment. Transfected cells were then infected with Ad.NFκBLuc 24 h prior to IL-1β treatment. NF-κB transcriptional activity was then assessed at 5 h following IL-1β stimulation by quantifying luciferase activity in 5 μg of protein lysate (mean ± SE; n = 6). *, Student's t test demonstrated a significant reduction (32%) in IL-1β-stimulated NF-κB activity in the presence of Nox2 siRNA compared to scrambled siRNA. The dotted line demonstrates the baseline NF-κB activity prior to IL-1β stimulation.