Neutrophils deficient in RhoA and RhoB are preactivated. (A) Genotypes and designations of mouse strains used in this study. (B) Immunoblots (IB) depicting RhoA, RhoB, and RhoC expression in neutrophils derived from the indicated mouse strains. Tubulin served as loading control and mouse brain lysate as RhoC control. (C) Cdc42 and Rac1 activity in unstimulated, Rho-deficient neutrophils in suspension. (D) Analysis of F-actin content of RhoB KO² and RhoA/B dKO neutrophils upon stimulation with fMLF and CXCL1. (E) F-actin distribution in RhoB KO² and RhoA/B dKO neutrophils stimulated with vehicle, fMLF, or CXCL1. The scale bar represents 5 µm. (F) Analysis of secondary granule exocytosis of RhoA KO, RhoB KO¹, and RhoA/B dKO neutrophils after treatment as indicated, displayed as lactoferrin release relative to unstimulated WT¹ neutrophils. (G) Phosphorylation of p38 MAPK in suspension neutrophils as indicated, stimulated with 10 ng/mL TNFα as indicated; total p38 served as control. (H-I) Surface expression of CD62L, CRAMP, and FPR on RhoB KO² and RhoA/B dKO neutrophils (H) or WT¹ neutrophils pretreated with inhibitors. Means ± SEM (n = 3): (D) *P < .05, **P < .01, unpaired 2-tailed Student t test; (F) *P < .05, **P < .01 by 2-way analysis of variance (ANOVA) post-hoc Bonferroni multiple comparison; (I) *P < .05, **P < .01 by 1-way ANOVA post-hoc Dunnett’s multiple comparison.