Integrin activation is dependent on WASp-mediated de novo actin polymerization but also on PIP₂ synthesis. (A–C) F-actin polymerization (A), soluble ICAM-1 binding (B), and LFA-1 clustering (C) of Was^−/− neutrophils reconstituted with WASp, vector control, or a WASp mutant lacking the VCA domain (WASpΔVCA) after stimulation with CXCL1 in solution. n = 3. (D and E) PIP₂ (D) and PIP₃ (E) levels of WT, Skap2^−/−, and Was^−/− neutrophils after CXCL1 stimulation in solution or plating on E-selectin with shear. n = 2. (F) Subcellular localization of talin-1 in unstimulated or CXCL1-stimulated WT neutrophils pretreated with DMSO, PAO, or Lat. A. Representative images and statistics of talin-1 plasma membrane localization are shown. 40 cells/experiment were analyzed. n = 3. (G) Immunoprecipitation of β₂ integrin in IL-8– or E-selectin–stimulated HL-60 cells after pretreatment with DMSO or PAO. Precipitates were immunoblotted with anti–talin-1 and anti–kindlin-3 or anti–β₂ integrin antibody. Input was immunoblotted with anti-GAPDH antibody. Quantification is shown below. n = 3. *, P < 0.05; one-way ANOVA (A and D–F), two-way ANOVA (B and C), or Student's t test (G). Data are means ± SEM. Ctrl, control; E-sel., E-selectin; IP, immunoprecipitate; MSCV, murine stem cell virus.