The interaction of Fyn with LAT is abolished by mutations in the 3 distal C-terminal tyrosines of LAT. (A) Platelets from WT (left panel) and Lat3YF (right panel) mice were stimulated by 5 nM Cvx for the time indicated, and LAT was immunoprecipitated and submitted to an in vitro kinase (IVK) assay in the presence of [γ-³²P]ATP. After separation by 10% SDS-PAGE, the radioactivity incorporated in LAT was assessed by PhosphorImager (PerkinElmer, Shelton, CT) analysis. (B) Alternatively, following immunoprecipitation with the anti-LAT antibody, immunoprecipitated complexes were dissociated and reimmunoprecipitated with anti-Src family antibody. After separation by 10% SDS-PAGE and transfer onto nitrocellulose, the reimmunoprecipitate was immunoblotted with a specific anti-Fyn antibody. (C) Fyn was immunoprecipitated from lysate of WT (left panel) or Lat3YF (right panel) platelets stimulated by 5 nM Cvx for the indicated times. Immunoprecipitated complexes were submitted to an in vitro kinase assay in the presence of [γ-³²P]ATP and reimmunoprecipitated with the anti-LAT antibody as described. The radioactivity incorporated in LAT was assessed by PhosphorImager analysis. Results are representative of 2 independent experiments.