Mechanism of Epac/Rap-induced increase in barrier function. (A) Immunofluorescence of fixed HUVECs treated for 30 minutes with F + R or O-Me-cAMP (100 μM) or control untreated cells (ctrl) using antibodies to AF-6 (top row), VE-cadherin (middle row), or FITC-phalloidin to detect actin filaments (bottom row). Scale bar = 10 μm. (B) Immunofluorescence of fixed HPAECs cultured in complete medium, low calcium medium, or after calcium replenishment (Plus Ca2⁺). Cells were treated with O-Me-cAMP for 30 minutes (+) or vehicle control (–) and fixed and stained for VE-cadherin (top row) or FITC-phalloidin to detect actin filaments (bottom row). Scale bar = 20 μm.