RASIP1 is a RAP1 effector that controls EC junction morphology. (A) Pull-down assay using purified RAP1A, RHOA, and RAC1 GST fusion proteins with control (c) or RASIP1 shRNA (kd) HUVEC lysates. RASIP1 interacts weakly with RAP1A-GST loaded with GDP, and more strongly with RAP1A loaded with nonhydrolyzable GTPγS. No significant interaction is seen with RHOA or RAC1. (B) RAP1 activation assay with control and RASIP1 shRNA-treated HUVEC lysates. No change in RAP1A activity is seen in the unstimulated (–cBiMPs) or activated (+cBiMPs) state. (C-H) Phalloidin (C,F, green in E,H) and VE-cadherin (D,G, red in E,H) staining in control (C-E) and RASIP1 (F-H) siRNA-transfected HUVEC treated with EGTA/cBiMPs. Note the diffuse and discontinuous appearance of VE-cadherin staining in panel G, and reduced F-actin incorporation at junctions in panel F. Scale bars: 20 μm. (I-J) Actin staining (green) in control and RASIP1 KD HUVECs. Arrows indicate regions of bundled actin at the cell periphery; arrowheads show areas of diffuse or perpendicular actin at free edges. Scale bars: 20 μm. (K) Quantification of bundled actin at the cell perimeter. Each data point is an experimental replicate; each experiment comprised of 8 images with numerous free edges. *P < .05 (Student t test).