Recovery of p85 and PI3K lipid kinase activity in anti-Ptyr immune complexes from thrombin receptor-activated platelets. Platelets were treated for 1 minute with either PBS vehicle or the indicated concentrations of SFLLRNPNDKY (SFLL) and lysed in 1% Triton-containing buffer. (A) Triton-solubilized protein was immunoprecipitated with either no antibody (lane 1) or with anti-Ptyr antibody (lanes 2 through 9). Immune complexes were washed and solubilized in SDS-PAGE sample buffer. Proteins were separated by SDS-PAGE and immunoblotted with anti-p85 monoclonal antibody. Molecular mass markers in kilodaltons are indicated at the left. IP; immunoprecipitation. (B) Triton-solubilized protein was immunoprecipitated with either no antibody (lane 1) or with anti-Ptyr antibody (lanes 2 through 9). Anti-Ptyr immune complexes were incubated with phosphatidylinositol (PI) and assayed for PI3K lipid kinase activity as described in Materials and Methods. Phosphorylated products were separated by TLC and visualized by autoradiography. The relative migration of PI4P standard (dotted oval) and PI3P are indicated at the right. The radioactivity (net counts) present in each spot was quantitated directly on the TLC plate and was as follows: lane 1, 9,713; lane 2, 11,216; lane 3, 13,226; lane 4, 13,324; lane 5, 7,995; lane 6, 4,830; lane 7, 18,036; lane 8, 24,355; lane 9, 29,064. Results are representative of several independent experiments. (C) Platelets were treated with the indicated concentrations of SFLLRNPNDKY (SFLL) and lysed. The Triton-solubilized lysates were immunoprecipitated with anti-Ptyr antibody in the absence (−) or presence (+) of excess (5 mmol/L) phosphotyrosine (Ptyr). The anti-Ptyr immune complexes were examined for PI3K activity as in (B).