Molecular partners in human platelets in complex with PEAR1. (A) Western blots of c-Src, Fyn, Lyn, p85 PI3K, and Syk after coimmunoprecipitation with PEAR1. Stirred platelets were stimulated or not with collagen and lysed at 2 minutes (30% aggregation); the immunoprecipitate by PEAR1-EC Ab is shown, together with total platelet lysate, in comparison to an IgG goat control antibody (C). (B) PEAR1 phosphorylation in platelets activated with PEAR1-EC Ab (1 μg/mL) after 3 minutes, with or without stirring. (C) Western blot analysis of the kinetics of the phosphorylation status of SFK activation loop (SFK-P), c-Src, and Fyn phosphorylation in the inhibitory loop, PEAR1-P, PEAR1, Akt-P, and β3 for washed unstirred human platelets treated with apyrase (0.4 U/mL), aspirin (550μM) and eptifibatide (10 μg/mL) on platelet activation with PEAR1-EC Ab (7 μg/mL) for different times (0-10 minutes), as indicated. Right panel: quantitative imaging for the evolution of the phosphorylation status of SFK, c-Src, Fyn, PEAR1 and Akt after PEAR1-EC Ab stimulation with time (NS, nonspecific band). (D) Western blot analysis of the kinetics of recruitment of c-Src, Fyn, and p85 PI3K in the PEAR1 complex after coimmunoprecipitation with PEAR1-EC Ab. Unstirred platelets were stimulated with PEAR1-EC Ab (7 μg/mL) for different times (0-10 minutes), as indicated. Right panel: quantitative imaging for the evolution of the recruitment of c-Src, Fyn, and p85 PI3K after PEAR1-EC Ab stimulation. (E) Western blot analysis of the recruitment of c-Src, Fyn, and p85 PI3K in the PEAR1 complex after coimmunoprecipitation with PEAR1-EC Ab. Unstirred platelets were stimulated with PEAR1-EC Ab (7 μg/mL) for 5 minutes in the presence or the absence of PP1 (10μM). (F) Western blot analysis of the recruitment of c-Src and c-Src phosphorylated in the inhibitory loop in the PEAR1 complex after coimmunoprecipitation with PEAR1-EC Ab. Unstirred platelets were stimulated with PEAR1-EC Ab (7 μg/mL) for 5 minutes. Vertical lines indicate a repositioned gel lane.