Tyrosine phosphorylation of p72^SYK and p53/56^LYN in platelets activated by echicetin-IgMκ complex.

After activation, platelets were lysed by 1.2% Triton X-100 and cytoskleton was removed by centrifugation at 100 000g. Supernatant was used for immunoprecipitation by specific anti-p72^SYK (A) or anti-p53/56^LYN (B) antibodies coupled to protein A–Sepharose 4B. Immunoprecipitated proteins were eluted by 1% SDS, separated by SDS-PAGE, transferred to PVDF membrane, and stained with antiphosphotyrosine antibody (4G10) or with specific anti-p72^SYK and anti-p53/56^LYNantibodies. Proteins from cytoskeleton were solubilized in 1% SDS, separated by SDS-PAGE, transferred to PVDF membrane, and stained with anti-p53/56^LYN antibodies.