GPIb-stimulated tyrosine phosphorylation of LAT and PLCγ2 are absent in platelets lacking FcR γ-chain.

(A) Washed platelets from wild-type C57BL/6 mice were stimulated with 10 μg/mL vWF plus 0, 3, or 6 μg/mL botrocetin for 5 minutes. After lysis, platelets were immunoprecipitated with anti-PLCγ2 Ab. (B) Washed platelets from wild-type C57BL/6 and FcR γ-chain–deficient mice were stimulated with 6 μg/mL botrocetin plus 10 μg/mL vWF for the indicated time periods. After lysis, PLCγ2 (i) and LAT (ii) were immunoprecipitated with the corresponding Ab, respectively. The precipitated proteins in A and B were separated by SDS-PAGE and were analyzed by 4G10 plus PY20 immunoblotting. The loaded proteins were confirmed to be at comparable levels by immunoblotting with the corresponding Ab. The data are representative of 3 experiments. pY indicates phosphotyrosine.