Western blot validation of inverse GSK3A^Ser21, RASGRP2^Ser567phosphorylation, and RAP1 pull-down after stimulation with ADP, Iloprost, and ADP+Iloprost. (A) Human platelets (3 × 10⁸/mL) were stimulated by ADP (10 μM, 1 minute), Iloprost (2 nM, 1 minute), or both; notably, slightly different than for the phosphoproteomics data. After stimulation, platelets were lysed and subjected to western blot GSK3A^Ser21, RASGRP2^Ser567, and Rap1 pull-down assay. The representative blots of 3 independent experiments are shown. (B) Immunoblots using selected phosphospecific antibodies were done in triplicate and (C) quantified using the ImageJ program. The intensities of the phosphorylation signals were normalized to actin. Data are normalized against controls, results are given as means ± standard error of the mean (n = 3, + P < .05 compared with the control, *P < .05 compared with the Iloprost sample).