FcR γ-chain associated with Src and Lyn is required for tyrosine phosphorylation of Syk.

(A) Samples were prepared as described in the legend for Figure 2C. The precipitates with GST-Syk-SH2 from human platelet lysates were eluted with Glutathione Elution Buffer (10 mM glutathione in50 mM Tris/HCl, pH 8.0), and the resultant eluates were subjected to re-immunoprecipitation with anti-Src and anti-Lyn MoAbs, respectively. Precipitated proteins were separated by SDS-PAGE and were analyzed by immunoblotting with anti-FcR γ-chain (i and ii), anti-Src (i), and anti-Lyn (ii) Abs. (Bi) Washed platelets prepared from wild-type C57BL/6 and FcR γ-chain–deficient mice were stimulated with 6 μg/mL botrocetin plus 10 μg/mL vWF for 3 minutes. Syk was immunoprecipitated from the lysates, and precipitates were separated by SDS-PAGE and analyzed with 4G10 plus PY20 and anti-Syk immunoblotting. pY indicates phosphotyrosine. (Bii) Level of Syk tyrosine phosphorylation shown in Bi was quantified by densitometry. Columns and error bars represent means ± SEM (n = 3).