FIG. 5.

Rac1 recruits Nox2 into the endosomal compartment by associating with IL-1R1 and is required for efficient redox-dependent recruitment of TRAF6 to the endosomal IL-1R1/MyD88 complex. (A) MCF-7 cells were transfected with MyD88 siRNA, Rac1 siRNA, or a scrambled (Scr) siRNA 48 h prior to examining MyD88 or Rac1 expression by Western blotting. (B) MCF-7 cells were transfected with the indicated siRNAs, infected with Ad.NFκBLuc prior to IL-1β stimulation, and analyzed for luciferase activities at 6 h post-cytokine treatment (mean ± standard error [SE]; n = 3). Paired comparisons (*, †) demonstrate significant differences (P < 0.05). (C) MCF-7 cells were transfected with the indicated siRNAs prior to a 20-min IL-1β stimulation and analysis of NADPH-dependent ·O₂⁻ production in the peak Iodixanol vesicular fractions (mean ± SE; n = 3). Paired comparisons (*, †) demonstrate significant differences (P < 0.05). (D and E) MCF-7 cells were transfected with the indicated siRNAs prior to IL-1β stimulation for 20 min. IL-1R1 was then immunoprecipitated from cell lysates, followed by Western blotting for MyD88, Rac1, TRAF6, and IL-1R1. (F) MCF-7 cells were transfected with the indicated siRNAs and stimulated with IL-1β for 20 min, and PNS were generated. An enriched endomembrane fraction was generated by centrifugation of PNS at 100,000 × g for 2 h. The endomembrane pellets were collected and evaluated for MyD88, Rac1, Nox2, TRAF6, and IL-1R1 by Western blotting.