The ubiquitination of Syk in activated human platelets. (A) Syk was immunoprecipitated from RIPA-lysed platelets that had been activated with convulxin for the indicated times. The immunoprecipitates were separated by SDS-PAGE and transferred to Immobilon. The blot was first probed for ubiquitin (antibody P4D1; Ai). This blot was stripped and probed for Syk (Aii). The same samples were run on another gel and transferred to Immobilon and probed for phosphotyrosine (Aiii). (B) Syk was immunoprecipitated from lysates of activated platelets with thrombin (T, lanes 2-4), thrombin plus SC57101 (T+, lanes 6-8), or convulxin (column 9) and probed for phosphotyrosine. (C) Platelets were preincubated for 10 minutes with 20 μM PP2 as indicated prior to activation with convulxin. Syk was immunoprecipitated and the blots were probed either for Syk (Ci) or for phosphotyrosine (Cii).