Integrin/Rap-dependent proadhesive phenotype and enhanced random migration in Rho-deficient neutrophils. (A) Cell surface expression of CD29, CD18, CD11a, CD11b, and CEACAM-1 on RhoB KO² and RhoA/B dKO neutrophils. (B) Flow cytometry analysis of ICAM1/Fc binding to unstimulated or PMA-stimulated RhoB KO² and RhoA/B dKO neutrophils. (C) Rap GTPase activation in WT¹, RhoB KO², and RhoA/B dKO neutrophils in suspension (S) or adhered to fibronectin (A). Total Rap served as control. (D) RIAM translocation analyzed in membrane (Mem) and cytosol (Cyto) fractions derived from unstimulated or fMLF-stimulated RhoB KO² or RhoA/B dKO neutrophils. (E) Cell surface expression of Plexin-B1 in RhoB KO² and RhoA/B dKO neutrophils. (F-G) Static adhesion assay comparing (F) neutrophils of indicated genotypes and (G) WT neutrophils with and without Tat-C3 pretreatment. (H) Quantification of uropod retraction defects during haptotaxis expressed as percentage of neutrophils displaying trailing uropods (n = 4; images represent 4 independent fields per experiment). The scale bar represents 10 µm. (I) Neutrophil tracking during random migration (left, n = 3), quantification of total distance migrated per neutrophil (right, ***P < .005 by 1-way ANOVA post hoc Dunnett’s multiple variance test). Means ± SEM (n = 3): (B,F,G,H) *P < .05, **P < .01, ***P < .005, ****P < .001 by unpaired 2-tailed Student t test.