GPVI proximal signaling is defective in CD148-deficient platelets. (A) Whole-cell lysates (WCLs) prepared of resting and collagen-related peptide (CRP)–activated platelets from wild-type (WT) and CD148 transmembrane-knockout (KO) mice were Western blotted with an anti-phosphotyrosine antibody (p-Tyr). Platelets were stimulated with 10 μg/mL CRP for 90 and 300 seconds (sec). Bands corresponding to Src family kinases (SFKs) and FcR γ-chain are indicated. (B) Syk and (C) PLCγ2 were immunoprecipitated from equal amounts of WCLs and blotted with a anti-phosphotyrosine antibody. Membranes were subsequently stripped and reblotted with anti-Syk and anti-PLCγ2 antibodies. (Di-vi) WCLs prepared of platelets stimulated for 0, 30, and 90 seconds with CRP were Western blotted for (Di) Lyn p-Tyr-507, (Diii) Src p-Tyr-529, and (Dv) SFK activation loop p-Tyr. Blots are representative of 4 to 6 experiments. (Dii, iv, vi) Band intensities were quantified from 4 separate experiments (mean ± SEM; *P < .05, **P < .01).