Epac/Rap1 regulation of TSP1. (A) Regulation of TSP1 and Id1 protein expression. HMVECs were treated with VEGF (10 ng/mL), 8CPT (100μM), VEGF, and 8CPT, or mock treated. After Western blot analysis (top), quantitation was performed using LI-COR software analysis (bottom). (B) Regulation of TSP1 protein expression after depletion of Epac1 and 2. HMVECs depleted of Epac1 or Epac2 by siRNA transfection (supplemental Figure 2) were treated with 8CPT (100μM) or mock treated. After Western blot analysis (top), quantitation was performed as above (bottom). (C) TSP1 protein expression after expression of CA Rap1 or Rap1GAP in HMVECs using lentiviral transduction. Cells were treated with 8CPT (100μM) or mock treated. After Western blot analysis (left), quantitation was performed as above (right). Representative experiments are shown.