The Skap2/WASp complex triggers actin polymerization, thereby enabling β₂ integrin activation by recruitment of talin-1 and kindlin-3. (A and B) F-actin polymerization of WT, Skap2^−/−, Was^−/−, and Skap2^−/−Was^−/− neutrophils (A) and Skap2^−/− neutrophils reconstituted with vector control, Skap2, or Skap2 W336K (B) after stimulation with CXCL1 in solution. n = 3. (C and D) Soluble ICAM-1 binding (C) and LFA-clustering (D) of CXCL1-stimulated WT, Skap2^−/−, and Was^−/− neutrophils after pretreatment with DMSO or Lat. A. For clustering, 50 cells/experiment were analyzed. n = 3. (E) Knockdown of WASp in HL-60 cells. Quantification is shown on the right. n = 3. (F–I) Control, Skap2, or WASp knockdown HL60 cells were pretreated with DMSO or Lat. A and left unstimulated, plated on E-selectin with shear, or stimulated with IL-8 in solution. Lysates were incubated with GST fusion proteins of the β₂ integrin cytoplasmic domain (F and G) or immunoprecipitated with anti–β₂ integrin antibody (H and I). Precipitates were immunoblotted with anti–talin-1 and anti–kindlin-3 or anti–β₂ integrin antibody, respectively. Input was immunoblotted with anti-GAPDH antibody. Quantifications are shown below. n = 3. *, P < 0.001; ^#, P < 0.05 versus all other groups; one-way ANOVA (A–B and F–I), two-way ANOVA (C and D), or Student's t test (E). Data are means ± SEM. Ctrl, control; E-Sel., E-selectin; IP, immunoprecipitate; PD, precipitate.