Fig. 3.
Influence of PI 3-kinase activity on Btk and Tec phosphorylation.
Aliquots of platelets were treated with 0.1% DMSO (vehicle for LY294002) or 50 μmol/L LY294002 for 10 minutes. Then 50 μg/mL collagen was added for 5 minutes, as indicated. (A) Btk or (B) Tec were purified from the soluble extracts by immunoprecipitation as described above. The proteins were analyzed by immunoblot with 4G10 total antiphosphotyrosine (top panels) and anti-Btk or anti-Tec antibody (bottom panels). Collagen-stimulated Btk phosphorylation and coprecipitation of phosphoproteins was inhibited by low-dose (5 μmol/L) LY294 002, (C) 10 nmol/L wortmannin, and (D) 100 nmol/L wortmannin. MgCl2 was added to the platelet extracts in (C) and (D).
