Modulation of PKA activity by PKC. (A) Freshly isolated PBLs were stimulated as described in Figure 1B, using PHA (4 μg/mL) and PMA (5 nM) as the stimuli, then analyzed for VASP-phospho-Ser157 by flow cytometry. MFI of phospho-VASP staining was normalized to PMA-induced phospho-VASP = 100% and H89 only-treated cells as 0%, with mean ± SD reported (n = 4). ^*P < .05 for difference versus PHA-stimulated; †P < .05 for inhibition by H89. (B) Fresh PBLs were treated for 20 minutes with vehicle or Bis I (10 μM), then each was stimulated 30 minutes with vehicle, PMA (2 nM), PHA (4 μg/mL), or PGE₂ (1 μM). Cells were then analyzed for VASP-phospho-Ser157 by flow cytometry. MFI of phospho-VASP staining was normalized to PGE₂-induced phospho-VASP = 100% and Bis I only-treated cells as 0%, with mean ± SD reported (n = 5). ^*P < .05 for effect of Bis I. (C) Fresh PBLs were treated 10 minutes with vehicle or Bis I (10 μM), then stimulated 30 minutes with vehicle, PGE₂ (1 μM), ISO (10 μM), forskolin (50 μM), or db-cAMP (200 μM). Cells were analyzed for VASP-phospho-Ser157 by flow cytometry, and net MFI values (MFI from vehicle-stimulated condition subtracted) is reported for the 2 PBL samples tested.