Time dependence of tyrosine phosphorylation in proteins from platelets activated by RANTES, TARC, or ADP and immunoprecipitation of PLCγ2.
(A) Washed platelets (5 × 108) were activated with 100 nmol/L RANTES, TARC, or 10 μmol/L ADP, respectively. Aliquots were removed at times indicated and dissolved in SDS containing inhibitors. After separation by SDS-PAGE and transfer to PDVF membranes the proteins were incubated with the antiphosphotyrosine antibody 4G10 before detection by chemiluminescence. Proteins with increased tyrosine phosphorylation are indicated by arrows on the left and identified phosphoproteins are indicated on the right. The results shown here are typical of at least 3 experiments performed with these doses and chemokines but analogous experiments with other chemokines interacting with platelets also gave similar results. (B) Aliquots of 108 platelets from points on a time range activated with TARC (50 nmol/L) solubilized in RIPA buffer were immunoprecipitated with antibodies against PLCγ2. After SDS-PAGE and Western blotting, the proteins were detected with 4G10 antiphosphotyrosine antibody. The membranes were stripped and treated with anti-PLCγ2antibodies.
