Protein phosphorylation in GP VI-stimulated wild-type and PI3K p85α—/— platelets. Murine platelets were treated using 0.2 mM acetylsalicylic acid and suspended in modified Tyrode-HEPES buffer containing 0.4 U/mL apyrase, 1 mM RGDS peptide, and 1 mM EGTA. The platelets were stimulated with CRP at 0, 1, and 5 μg/mL on an aggregometer with constant stirring and were lysed 90 seconds after stimulation. Then they were subjected to immunoprecipitation using anti-Syk (A), anti-LAT (B), anti-SLP-76 (C), anti-Btk (D), anti-Tec (E), anti-Akt (F), and anti-PLCγ2 (G) antibodies. Proteins were resolved using SDS-PAGE, transferred to nitrocellulose membrane, immunoblotted with antiphosphotyrosine (P-Tyr) antibody 4G10 or anti-phospho-Akt-specific antibody, and reprobed with the antibodies used for immunoprecipitation to demonstrate equal amounts of immunoprecipitated proteins in each lane. Identical results were obtained in at least 4 separate experiments.