Figure 6.
Figure 6. The Skap2 SH3 domain mediates WASp localization and integrin activation. (A and B) Subcellular localization of Skap2 and WASp in unstimulated or CXCL1-stimulated WT or Skap2−/− neutrophils. (A) Representative microscopy images. (B) Statistics of WASp plasma membrane localization. 75 cells/experiment were analyzed. n = 3. (C) Expression of Skap2 in WT, Skap2−/−, and Skap2−/− neutrophils reconstituted with Skap2, vector control, Skap2 R140M, or Skap2ΔSH3. Lysates were immunoblotted with anti-Skap2 and anti–α-tubulin antibody. n = 2. (D) Subcellular localization of Skap2 and WASp in unstimulated or CXCL1-stimulated Skap2−/− neutrophils reconstituted with Skap2, Skap2ΔSH3, or Skap2 R140M. n = 3. (E) Statistics of WASp plasma membrane localization. 75 cells/experiment were analyzed. n = 3. (F) Phosflow analysis of pAKT in Skap2−/− neutrophils reconstituted with the vector control, Skap2, or Skap2 R140M after stimulation with CXCL1. Fold-increase in pAKT compared with the unstimulated control is shown. n = 2. (G–I) IVM of TNF-inflamed postcapillary venules of WT mice after bone marrow transplantation of Skap2−/− hematopoietic stem cells retrovirally transduced with the vector control, Skap2, or Skap2ΔSH3. Rolling velocity (G), number of adherent cells (H), and number of extravasated cells (I) 2 h after TNF application are shown. n = 3 mice/group. (J) Soluble ICAM-1 binding of CXCL1-stimulated transduced Skap2−/− neutrophils. n = 3. (K) Expression of Skap2 in WT, Skap2−/−, and Skap2−/− neutrophils reconstituted with Skap2, vector control, or Skap2 W336K. Lysates were immunoblotted with anti-Skap2 and anti–α-tubulin antibody. n = 2. The same experiment as in C is shown. (L–N) IVM of TNF-inflamed postcapillary venules of WT mice after bone marrow transplantation of Skap2−/− hematopoietic stem cells retrovirally transduced with the vector control, Skap2, or Skap2 W336K. Rolling velocity (L), number of adherent cells (M), and number of extravasated cells (N) 2 h after TNF application are shown. n = 3 mice/group. (O) Soluble ICAM-1 binding of CXCL1-stimulated reconstituted Skap2−/− neutrophils. n = 3. *, P < 0.05; **, P < 0.01; Student's t test (B), one-way ANOVA (E–I and L–N), or two-way ANOVA (J and O). Data are means ± SEM. Ctrl, control; MSCV, murine stem cell virus.

The Skap2 SH3 domain mediates WASp localization and integrin activation. (A and B) Subcellular localization of Skap2 and WASp in unstimulated or CXCL1-stimulated WT or Skap2−/− neutrophils. (A) Representative microscopy images. (B) Statistics of WASp plasma membrane localization. 75 cells/experiment were analyzed. n = 3. (C) Expression of Skap2 in WT, Skap2−/−, and Skap2−/− neutrophils reconstituted with Skap2, vector control, Skap2 R140M, or Skap2ΔSH3. Lysates were immunoblotted with anti-Skap2 and anti–α-tubulin antibody. n = 2. (D) Subcellular localization of Skap2 and WASp in unstimulated or CXCL1-stimulated Skap2−/− neutrophils reconstituted with Skap2, Skap2ΔSH3, or Skap2 R140M. n = 3. (E) Statistics of WASp plasma membrane localization. 75 cells/experiment were analyzed. n = 3. (F) Phosflow analysis of pAKT in Skap2−/− neutrophils reconstituted with the vector control, Skap2, or Skap2 R140M after stimulation with CXCL1. Fold-increase in pAKT compared with the unstimulated control is shown. n = 2. (G–I) IVM of TNF-inflamed postcapillary venules of WT mice after bone marrow transplantation of Skap2−/− hematopoietic stem cells retrovirally transduced with the vector control, Skap2, or Skap2ΔSH3. Rolling velocity (G), number of adherent cells (H), and number of extravasated cells (I) 2 h after TNF application are shown. n = 3 mice/group. (J) Soluble ICAM-1 binding of CXCL1-stimulated transduced Skap2−/− neutrophils. n = 3. (K) Expression of Skap2 in WT, Skap2−/−, and Skap2−/− neutrophils reconstituted with Skap2, vector control, or Skap2 W336K. Lysates were immunoblotted with anti-Skap2 and anti–α-tubulin antibody. n = 2. The same experiment as in C is shown. (L–N) IVM of TNF-inflamed postcapillary venules of WT mice after bone marrow transplantation of Skap2−/− hematopoietic stem cells retrovirally transduced with the vector control, Skap2, or Skap2 W336K. Rolling velocity (L), number of adherent cells (M), and number of extravasated cells (N) 2 h after TNF application are shown. n = 3 mice/group. (O) Soluble ICAM-1 binding of CXCL1-stimulated reconstituted Skap2−/− neutrophils. n = 3. *, P < 0.05; **, P < 0.01; Student's t test (B), one-way ANOVA (E–I and L–N), or two-way ANOVA (J and O). Data are means ± SEM. Ctrl, control; MSCV, murine stem cell virus.

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