E-selectin–mediated slow rolling and GPCR-dependent adhesion are regulated by Skap2. (A and B) E-selectin–mediated slow rolling of WT and Skap2^−/− neutrophils. (A) In vivo rolling velocities in TNF-inflamed postcapillary venules treated with pertussis toxin and blocking anti–P-selectin antibody. (Inset) Cumulative histogram data in a bar graph are shown. n = 4 mice/group. (B) In vitro rolling velocities in autoperfused flow chambers coated with E-selectin or E-selectin and ICAM-1. n = 4 mice/group. (C) Knockdown of Skap2 in HL-60 cells. Quantification is shown on the right. n = 3. (D) Adhesion of control or Skap2 knockdown HL60 cells on flow chambers coated with E-selectin and an isotype, anti–β₂ integrin conformation reporter (KIM127), or anti–β₂ integrin (TS2/4) antibody. n = 3. (E and F) Adhesion of neutrophils in postcapillary venules of WT and Skap2^−/− mice after i.v. injection of CXCL1 (E) or LTB₄ (F). n = 4 mice/group. (G and H) IVM of postcapillary venules of WT and Skap2^−/− mice before or 1 h after superfusion with CXCL2. Number of adherent cells (G) and number of extravasated cells (H) are shown. n = 4 mice/group. (I) Adhesion of WT and Skap2^−/− neutrophils on autoperfused flow chambers coated with P-selectin/ICAM-1 or P-selectin/ICAM-1 and CXCL1 or PMA. n = 3 mice/group. (J) Soluble ICAM-1 binding of CXCL1-, PMA-, or Mn²⁺-stimulated WT and Skap2^−/− neutrophils. n = 3. (K) Adhesion of control or Skap2 knockdown HL60 cells on flow chambers coated with P-selectin, IL-8, and an isotype, anti–β₂ integrin conformation reporter (mAb24), or anti–β₂ integrin (TS2/4) antibody. n = 3. (L) Soluble ICAM-1 binding of IL-8–stimulated control or Skap2 knockdown HL-60 cells. n = 3. (M and N) LFA-1 clustering on WT and Skap2^−/− neutrophils. (M) Representative microscopy images. (N) Percentage of cells showing LFA-1 clusters after CXCL1 stimulation in solution or after plating on E-selectin with shear. 50 cells/experiment were analyzed. n = 3. (O–Q) WT and Skap2^−/− neutrophils were stimulated with CXCL1 for 3 min in solution, and lysates were immunoblotted with anti–p-ERK1/2 and anti-ERK1/2 (O), anti–p-p38 and anti-p38 (P), or anti–p-Akt, anti-Akt, and anti–α-tubulin antibody (Q). Quantification is shown on the right. n = 3. (R) Concentration of intracellular calcium measured in Indo-1–labeled WT and Skap2^−/− neutrophils before and after CXCL1 stimulation. n = 3. *, P < 0.05; **, P < 0.001; ***, P < 0.001; ^#, P < 0.05 versus all time points; Student's t test. Data are means ± SEM. Ctrl, control; E-Sel., E-selectin; P-sel., P-selectin.