Figure 5.
Figure 5. Cross talk of Skap2 and WASp is indispensable for β2 integrin activation and neutrophil recruitment. (A–C) IVM of TNF-inflamed postcapillary venules of WT, Was−/−, and Skap2−/−Was−/− mice. Rolling velocities (A), number of adherent cells (B), and number of extravasated cells (C) 2 h after TNF application are shown. n = 4 mice/group. (D) Adhesion of neutrophils in postcapillary venules of WT, Was−/−, and Skap2−/−Was−/− mice after i.v. injection of CXCL1. n = 4 mice/group. (E and F) Soluble ICAM-1 binding (E) and soluble fibrinogen binding (F) of CXCL1-stimulated WT, Was−/−, and Skap2−/−Was−/− neutrophils. n = 3. (G) Immunoprecipitation of Skap2 in unstimulated WT neutrophils or neutrophils plated on E-selectin with shear or stimulated with CXCL1 in solution. Immunoprecipitates were immunoblotted with anti-WASp and anti-Skap2 antibody. Input was with anti–α-tubulin antibody. n = 3. (H) WT neutrophils were left unstimulated, plated on E-selectin with shear, or stimulated with CXCL1 in solution. Lysates were incubated with GST alone (control), GST fusion proteins of the Skap2 CC, PH, or SH3 domains, or full-length Skap2. Precipitates were immunoblotted with anti-WASp, and input was with anti–α-tubulin antibody. n = 3. (I and J) In vitro co-purification of His-WASp by different Skap2 GST fusion proteins. Precipitates were immunoblotted with anti-His or anti-GST, and input controls were with anti-GST antibody. n = 3. (K–N) WT and Skap2−/− or Was−/− neutrophils were left unstimulated, plated on E-selectin with shear, or stimulated with CXCL1 in solution. Lysates were immunoprecipitated with anti-WASp (K and L) or anti-Skap2 (M and N) antibody followed by immunoblotting with anti-phosphotyrosine (4G10), anti-WASp, or anti-Skap2 antibody. Input was immunoblotted with anti–α-tubulin antibody. Quantification is shown on the right. n = 3. *, P < 0.05; #, P < 0.05 versus all time points; Student's t test (A–D and K–N) or one-way ANOVA (E and F). Data are means ± SEM. Ctrl, control; E-Sel., E-selectin; IP, immunoprecipitate; PD, precipitate.

Cross talk of Skap2 and WASp is indispensable for β2 integrin activation and neutrophil recruitment. (A–C) IVM of TNF-inflamed postcapillary venules of WT, Was−/−, and Skap2−/−Was−/− mice. Rolling velocities (A), number of adherent cells (B), and number of extravasated cells (C) 2 h after TNF application are shown. n = 4 mice/group. (D) Adhesion of neutrophils in postcapillary venules of WT, Was−/−, and Skap2−/−Was−/− mice after i.v. injection of CXCL1. n = 4 mice/group. (E and F) Soluble ICAM-1 binding (E) and soluble fibrinogen binding (F) of CXCL1-stimulated WT, Was−/−, and Skap2−/−Was−/− neutrophils. n = 3. (G) Immunoprecipitation of Skap2 in unstimulated WT neutrophils or neutrophils plated on E-selectin with shear or stimulated with CXCL1 in solution. Immunoprecipitates were immunoblotted with anti-WASp and anti-Skap2 antibody. Input was with anti–α-tubulin antibody. n = 3. (H) WT neutrophils were left unstimulated, plated on E-selectin with shear, or stimulated with CXCL1 in solution. Lysates were incubated with GST alone (control), GST fusion proteins of the Skap2 CC, PH, or SH3 domains, or full-length Skap2. Precipitates were immunoblotted with anti-WASp, and input was with anti–α-tubulin antibody. n = 3. (I and J) In vitro co-purification of His-WASp by different Skap2 GST fusion proteins. Precipitates were immunoblotted with anti-His or anti-GST, and input controls were with anti-GST antibody. n = 3. (K–N) WT and Skap2−/− or Was−/− neutrophils were left unstimulated, plated on E-selectin with shear, or stimulated with CXCL1 in solution. Lysates were immunoprecipitated with anti-WASp (K and L) or anti-Skap2 (M and N) antibody followed by immunoblotting with anti-phosphotyrosine (4G10), anti-WASp, or anti-Skap2 antibody. Input was immunoblotted with anti–α-tubulin antibody. Quantification is shown on the right. n = 3. *, P < 0.05; #, P < 0.05 versus all time points; Student's t test (A–D and K–N) or one-way ANOVA (E and F). Data are means ± SEM. Ctrl, control; E-Sel., E-selectin; IP, immunoprecipitate; PD, precipitate.

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