Figure 3.

Aberrant tyrosine phosphorylation in Shp1- and Shp2-deficient platelets. (A) (i) Whole cell lysates (WCLs) from Shp1 KO and Shp1 WT were western blotted with anti-GPVI antibody that recognize both intact GPVI and the sheddase-generated C-terminal tail, with anti-FcRγ chain and α-tubulin antibodies. (i) Representative western blots from n = 3 independent experiments and (ii-iv) quantification of intact GPVI, GPVI tail, and FcRγ chain are shown. Data are mean ± SEM, **P < .01. (B) WCLs of resting and CRP-stimulated platelets from Shp1 KO and Shp1 WT mice were western blotted with anti-phosphotyrosine (p-Tyr), -Src p-Tyr418, -Syk p-Tyr519/520, and -PLCγ2p-Tyr1217 antibodies. Membranes were stripped and reblotted with anti-pan Syk, pan PLCγ2, and actin antibodies. Representative blots and densitometry quantification from n = 3-7 independent experiments/genotype (mean ± SEM; *P < .05, **P < .01, ***P < .001). (C) WCLs of resting and CRP-stimulated platelets from Shp2 KO and Shp2 WT mice were western blotted with anti-p-Tyr antibody, stripped, and reblotted with anti-actin antibody. Representative blots from n = 5 independent experiments. (D-E) WCLs of washed platelets from bovine serum albumin (BSA) nonadherent and fibrinogen (fib)-adhered (D) Shp1 KO, (E) Shp2 KO, and litter-matched WT mice in the presence of 10 μM indomethacin and 2 U/mL apyrase were western blotted with anti-p-Tyr and -Src p-Tyr418 antibodies. (E top panels) Equal amounts of total protein were resolved on the same membrane, but different exposure times are shown to highlight differentially phosphorylated proteins in BSA nonadherent and fib-adhered platelets. Syk and PLCγ2 were immunoprecipitated (IP) from WCLs and western blotted with anti-p-Tyr antibody. Membranes were reblotted with anti-pan Syk and -pan PLCγ2 antibodies. Representative blots and densitometry quantification of n = 3 independent experiments/genotype (mean ± SEM; *P < .05).