EPAC1-RAP1–dependent junctional localization and activity of RASIP1. (A,C,F) Representative images of HUVECs transfected with the indicated siRNA were stained for RASIP1 (red) + β-catenin (green) + DAPI (blue) (A), VE-cadherin (red) + phalloidin for F-actin (green) + DAPI (blue) (C), or nmMHCIIB (red) + β-catenin (green) + DAPI (blue) (F). VE-cadherin in panel C was stained prior to permeabilization so as to capture surface signal only. (B) Loss of RAP1A+B or EPAC1 significantly reduced RASIP1 localization to cell-cell contacts. (D) Line scans perpendicular to cell-cell junctions were performed on images similar to those shown in panel C; VE-cadherin (red lines) and F-actin (green lines) signal intensities vs scan position are depicted in these representative scan data. (E) Distance between the highest peaks of F-actin signal (green arrowheads in panel D) and VE-cadherin signal (red arrows in panel D) were measured and plotted against treatment conditions. (G) Cell-cell junctions that are positive for nmMHCIIB (solid white arrows in panel F) are frequently found in control cells, whereas junctions devoid of nmMHCIIB (open white arrows in panel F) were abundant in RASIP1, RAP1A+B, and EPAC1 siRNA-treated cells. All graphs summarize data from 3 independent experiments; each data point represents mean values from 1 experiment. Thirty junctions were evaluated per condition in each experiment. Red asterisks denote statistically significant difference between the indicated groups vs control. P < .01 (unpaired Student t test). Scale bars: 10 μm. All error bars are interexperimental SEM.