Crucial role of Syk in platelet activation downstream of CLEC-2. (A) Washed murine platelets were stimulated with 50 nM rhodocytin for the indicated times. Whole-cell lysates or immunoprecipitates with antibodies against PLCγ2, Vav1, or Vav3 were separated by SDS-PAGE and Western blotted with the indicated antibodies. (B) Control or Syk-deficient platelets were stimulated with 30 nM rhodocytin and platelet aggregation was monitored using an aggregometer. (Ci-ii) Washed human platelets, pretreated with or without 30 μM PP2, were stimulated with 50 nM rhodocytin or 10 μg/mL convulxin for the indicated times. Reactions were terminated by addition of an equal volume of 2 × lysis buffer. Platelet lysates were precleared and detergent-insoluble debris was clarified by centrifugation. The resultant supernatant was incubated with 40 μL glutathione beads associated with GST fusion protein containing tandem Syk SH2 domains. Precipitated proteins were separated by SDS-PAGE and Western blotted with an antibody to CLEC-2. (Ciii) CLEC-2 associated with 10 μg of CLEC-2 phospho-YXXL–containing peptide plus avidin-sepharose was detected by CLEC-2 antibody. The data are representative of 2 to 5 experiments.