Time course of PKA substrate phosphorylation in iloprost-stimulated human platelets. Washed human platelets (3 × 10⁸/mL) were incubated with iloprost (2nM) for the indicated time and processed for western blot analysis with antibodies directed against (A) Phospho-PKA substrates (RRXpS/T), (B) P-GRP2^Ser587, P-GSK3α^Ser21, P-GSK3β^Ser9, P-LASP^Ser146, P-VASP^Ser157, P-VASP^Ser239, or total GRP2 (CalDAG-GEFI) antibodies. (C) Immunoblots were scanned and intensities of the phospho-specific antibodies were normalized to the total GRP2 signal. All images were quantified by the Image J program and expressed as fold changes, where control samples were taken as 1. Results are mean ± SEM, n = 4. ⁺P < .05 compared with the control; *P < .05 compared with 15-second samples.