Csk regulates ITAM-, ITIM-, and integrin-dependent signaling in Csk^ASplatelets. (A-E) Platelets from Csk^AS mice were pretreated with either dimethyl sulfoxide (DMSO) or 3-IB-PP1 (10 μM, 10 minutes). (Ai-Aiv) Representative aggregation and ATP secretion traces of platelets from Csk^AS mice in response to the indicated agonists (n = 3-4 mice per condition per genotype). (Bi) Representative DIC microscopy images of resting (basal) and thrombin-stimulated (0.1 U/mL, 5 minutes) platelets spread on fibrinogen-coated cover-slips (100 μg/mL, 45 minutes, 37°C; scale bar, 5 μm). (Bii) Mean surface area of individual platelets quantified by ImageJ (n = 256 platelets per condition). (C-D) Lysates of platelets incubated with or without CRP (30 μg/mL, C) or CLEC-2 antibody (10 μg/mL, D) for the indicated times were blotted for p-Tyr and actin (Ci,Di) or analyzed by capillary-based immunoassays as described in Figure 5A (Cii-Ciii,Dii-Diii) (n = 3-4 mice per genotype). See also supplemental Figure 10A-B. (E) Increased G6b-B phosphorylation and assembly of the G6b-B-Shp1-Shp2 complex in collagen-stimulated Csk^AS platelets treated with 3-IB-PP1. Lysates of basal and collagen-stimulated (30 μg/mL, 90 seconds) platelets were immunoprecipitated with anti-G6b-B antibody and blotted for p-Tyr, G6b-B, Shp1, and Shp2. *P < .05, **P < .01, ***P < .001; ordinary (B) or repeated-measures (C-D) 2-way ANOVA with Sidak’s test; data represent mean ± SEM.