FIG. 2.

IL-1β stimulates endosomal NADPH-dependent ·O₂⁻ production required for TRAF6 recruitment. (A) MCF-7 cell vesicular fractionation on Iodixanol gradients was performed following treatment with IL-1β and biotin-transferrin for 20 min. Each fraction was evaluated by Western blotting for the various molecular markers as indicated. (B) NADPH-dependent ·O₂⁻ production following IL-1β stimulation was assessed in each Iodixanol fraction in a lucigenin-based chemiluminescence assay. (C) MCF-7 cell vesicular fractions from SOD(m) (bovine Cu/ZnSOD protein was added to the cellular medium prior to vesicular isolation), SOD(v) (bovine Cu/ZnSOD was added to vesicular fractions following isolation), and control (Ctrl; no Cu/ZnSOD addition) treatment conditions were incubated with PBS, pronase, or pronase plus Triton X-100 (0.5%) at 37°C for 30 min. The samples were then separated by SDS-PAGE and analyzed by Western blotting using an anti-Cu/ZnSOD antibody. hSOD, human Cu/ZnSOD; bSOD, bovine Cu/ZnSOD. (D) MCF-7 cells were loaded with purified bovine Cu/ZnSOD at the time of cytokine treatment, and NADPH-dependent ·O₂⁻ production in the peak vesicular fractions (no. 2 to 4) was evaluated using lucigenin in the presence or absence of 50 μM KCN (Cu/ZnSOD inhibitor) (mean ± SE; n = 3). (E) ·O₂⁻ production in the peak vesicular fractions was evaluated by ESR using the spin-trap DMPO in the presence and absence of 100 μM of NADPH (conditions 1 to 4). Assays were also performed with isolated vesicular fractions loaded with Cu/ZnSOD or catalase proteins (conditions 5 to 7). Asterisks mark DMPO/^·OH adducts. The y axis represents 5 × 10⁴ arbitrary units of intensity, and the x axis represents the magnetic field in Gauss. (F) Pooled vesicular fractions (2 to 4) were evaluated for NADPH-dependent ·O₂⁻ production in the absence or presence of DPI (Nox inhibitor) or rotenone (mitochondrial respiratory chain complex I inhibitor). (G) The vesicular fractions (2 to 4) were evaluated for NADPH-dependent ·O₂⁻ production from cells infected with dynamin(K44A)-expressing or empty vector (Ad.BglII) recombinant adenovirus prior to IL-1β stimulation. (H) MCF-7 cells were loaded with Cu/ZnSOD/catalase protein and treated with IL-1β for 20 min. Whole-cell lysates or purified vesicular fractions were evaluated by Western blotting for MyD88 and TRAF6 (upper panel). Infrared quantification of MyD88 and TRAF6 band intensities from the vesicular fractions are plotted in the lower panel (mean ± SE; n = 3). *, Student's t test demonstrated a significant reduction in endosomal TRAF6 in the presence of Cu/ZnSOD/catalase loading (P < 0.05).