Figure 10. Rap1b regulates ECM... | Rockefeller University Press

Figure 10.

$Figure 10. Rap1b regulates ECM degradation via SHP-1. (A) Immunoblot analysis of SHP-1 activation; WCL from WT and Rap1b−/− neutrophils with or without stimulation were processed for immunoprecipitation with SHP-1 antibody. Blots were first probed with anti–phospho-tyrosine (p-Tyr) antibody followed by SHP-1 antibody. (B) Immunoblot analysis of SHP-1 association to DRM fractions of WT and Rap1b−/− neutrophils with or without stimulation that were probed with SHP-1 and β-actin antibodies. (C) Immunofluorescence analysis of SHP-1 and F-actin in WT and Rap1b−/− neutrophils in resting condition or upon stimulation with fMLP and plating on Fg. Bar, 10 µm. Histogram represents percentage of cells with SHP-1 enriched at the uropod (mean ± SD; n = 3 independent experiment; **, P < 0.01; using unpaired Student’s t test). (D) Effect of SHP-1/2 inhibitor NSC87877 on matrix degradation. Bar, 10 µm. Mean ± SD. n = 3 independent experiment. *, P < 0.05; ***, P < 0.001). (E) p-Akt level of WT neutrophils that were pretreated with NSC87877 (20 µM) for 20 min. Blots are representative of 3 independent experiments and densitometric analysis are cumulative of 3 experiments (A, B, and E). Mean ± SD. *, P < 0.05; **, P < 0.01; unpaired Student’s t test.$

Rap1b regulates ECM degradation via SHP-1. (A) Immunoblot analysis of SHP-1 activation; WCL from WT and Rap1b^−/− neutrophils with or without stimulation were processed for immunoprecipitation with SHP-1 antibody. Blots were first probed with anti–phospho-tyrosine (p-Tyr) antibody followed by SHP-1 antibody. (B) Immunoblot analysis of SHP-1 association to DRM fractions of WT and Rap1b^−/− neutrophils with or without stimulation that were probed with SHP-1 and β-actin antibodies. (C) Immunofluorescence analysis of SHP-1 and F-actin in WT and Rap1b^−/− neutrophils in resting condition or upon stimulation with fMLP and plating on Fg. Bar, 10 µm. Histogram represents percentage of cells with SHP-1 enriched at the uropod (mean ± SD; n = 3 independent experiment; **, P < 0.01; using unpaired Student’s t test). (D) Effect of SHP-1/2 inhibitor NSC87877 on matrix degradation. Bar, 10 µm. Mean ± SD. n = 3 independent experiment. *, P < 0.05; ***, P < 0.001). (E) p-Akt level of WT neutrophils that were pretreated with NSC87877 (20 µM) for 20 min. Blots are representative of 3 independent experiments and densitometric analysis are cumulative of 3 experiments (A, B, and E). Mean ± SD. *, P < 0.05; **, P < 0.01; unpaired Student’s t test.