Multiple agents promote VASP phosphorylation in a PKA-dependent manner. Freshly isolated PBLs were stimulated 30 minutes with vehicle, PGE₂ (1 μM), forskolin (50 μM), or ISO (10 μM). (A) Cells were then lysed in sample buffer and subsequently analyzed by SDS-PAGE/immunoblotting for VASP expression. (B) Freshly isolated PBLs were treated 20 minutes with vehicle or H89 (10 μM), followed by a 30-minute stimulation with vehicle, ISO (10 μM), PGE₂ (1 μM), or PMA (5 nM). PBLs were then analyzed for expression of VASP-phospho-Ser157 by flow cytometry. Histograms show expression of phospho-VASP (x-axis) in vehicle (top) or H89 (bottom) treated cells (open region) and cells with the indicated treatments (filled region). Background fluorescence from isotype control mAb was superimposable for all conditions (not shown). (C) Freshly isolated PBLs were pretreated 5 minutes with serial dilutions of ICI-118551, stimulated 30 minutes with 100 nM or 1 μM ISO, then analyzed for expression of VASP-phospho-Ser157 by flow cytometry. Histograms of phospho-VASP for ISO-stimulated PBLs are shown (top), and the percent inhibition of ISO-stimulated VASP phosphorylation by antagonist is reported as mean ± SD (bottom; n = 3). (D) Freshly isolated PBLs (n = 2) were treated 10 minutes with vehicle or Rp-Br-MB-cAMPs and Rp-8-CPT-cAMPs (100 μM each), followed by 30-minute stimulation with PGE₂ (1 μM) or ISO (10 μM). VASP-phospho-Ser157 expression was analyzed by flow cytometry, and raw MFI values (without background MFI subtracted) are reported.