Cc2−/− platelets show hyperphosphorylated proteins after GPVI and CLEC-2–selective agonists, CRP and Rhod, stimulation over time. (A) Platelets (3 × 108/mL) from wild-type and Cc2−/− mice were stimulated with 10 µg/mL of CRP at 37 °C for 3 minutes. Platelet lysate of 30 µg was then loaded onto a 10% (wt/vol) SDS-PAGE gel. Then western blotting was performed to measure tyrosine phosphorylation using 1:5000 of HRP-conjugated anti-phosphotyrosine RC20 antibody. A protein-loading control (bottom panel) blot was stripped and reprobed using anti-Erk-1/2 Ab for detection of Erk-1 and -2 antigens. The data shown are a representative blot of similar results for 3 independent experiments. (B) Tyrosine phosphorylation of PLCγ2, Src, and Syk was detected from platelet lysate after stimulation with 10 µg/mL of CRP vs resting at time 0 and 90 seconds. Immunoprecipitation of PLCγ2, Src, and Syk from platelet lysates was performed followed by immunoblotting to detect (a) p-PLCγ2, using 1:5000 of an HRP-conjugated anti-phosphotyrosine RC20 antibody, (b) p-Src, using 1:2000 of antiphospho Src and 1:20 000 of anti-rabbit, and (c) p-Syk, using 1:20 000 of an HRP-conjugated anti-phosphotyrosine 4G10 antibody. PLCγ2, Src, and Syk antigens (bottom panel; a-c) loading control were confirmed by reprobing with polyclonal anti-PLCγ2, anti-Src, and anti-Syk antibodies, respectively. The relative intensity of tyrosine-phosphorylated PLCγ2, Src, and Syk was quantified by ImageJ software, version 1.46r. The data shown are a representative blot of similar results for 3 independent experiments. (C) Tyrosine phosphorylation of PLCγ2 and Syk was detected from platelet lysate after stimulation with 1.2 µg/mL of Rhod vs resting at time 0 and 90 seconds as described in (B).
