Tyrosine phosphorylation of Syk and its association with FcR γ-chain, Src, and Lyn are dependent on Src kinase activity.

(A) After incubation with 0.25% DMSO as the vehicle control or 10 μM PP1 for 5 minutes at 37°C, human platelets were stimulated with 6 μg/mL botrocetin plus 10 μg/mL vWF for the indicated time periods. The lysates were immunoprecipitated with anti-Syk MoAb. Precipitated proteins were then separated by SDS-PAGE and immunoblotted with 4G10 plus PY20 and anti-Syk MoAb. (B) Precipitated proteins with anti-Syk MoAb obtained in A were separated by SDS-PAGE and analyzed by immunoblotting with anti-FcR γ-chain, anti-Src, anti-Lyn, and anti-Fyn Abs as indicated in the left of each panel. (C) After pretreatment with 0.25% DMSO (control), 10 μM PP1 or 20 μg/mL jararaca-GPIb-BP, human platelets were stimulated with vWF plus botrocetin. The lysates were precipitated with GST-Syk-SH2, and the precipitated proteins were separated by SDS-PAGE and analyzed by immunoblotting with anti-FcR γ-chain, anti-Src, anti-Lyn, anti-Fyn, and anti-Syk Abs. The data are representative of 3 experiments. The arrows represent the band derived from the heavy chains of IgG. pY indicates phosphotyrosine.