RhoA acts as a negative regulator of the FPR-mediated oxidative burst. (A-B) Superoxide production by suspension neutrophils stimulated with (A) 10 µM fMLF with and without TNFα priming (10 ng/mL) or with (B) 100 ng/mL phorbol 12-myristate 13-acetate (PMA). Arrows indicate the addition of stimulus; dark black lines indicate KO responses. (C) H₂O₂ production by adherent, fMLF-stimulated neutrophils. Stimulation at t = 0. Dark black lines indicate KO responses. (D) Immunoblot of p47^phox and p22^phox in cytosolic (C) and membrane (M) fractions derived from WT² neutrophils stimulated with fMLF for 5 minutes and 10 ng/mL TNFα for 30 minutes, followed by fMLF for 5 minutes, or with PMA for 10 minutes. (E) Immunoblot of p47^phox, p22^phox, and PKCζ in membrane fractions derived from TNFα-primed or nonprimed RhoB KO² or RhoA/B dKO neutrophils stimulated with fMLF for 5 minutes. (F) Time course of p47^phox phosphorylation during fMLF stimulation of TNFα-primed WT¹ neutrophils. (G) Comparison of fMLF-induced (30s) p47^phox phosphorylation in RhoB KO² or RhoA/B dKO neutrophils in the presence or absence of TNFα priming. (H) Characterization of p47^phox Ser316 and Ser329 phosphorylation in WT¹ neutrophils after stimulation with 10 ng/mL TNFα or 100 ng/mL PMA. (I) Erk1/2 phosphorylation in Rho-deficient suspension neutrophils stimulated with TNFα or fMLF as indicated; total Erk served as control. Means ± SEM (n = 3): (C) *P < .05, **P < .01; unpaired 2-tailed Student t test.