Altered angiogenic cell signaling in Rap1-silenced endothelial cells. Endothelial cells transfected with siRNAs targeted against Rap1a/Rap1b or scrambled siRNA. (A) After 48 hours, HUVECs were serum-starved for 3 hours and then left untreated or stimulated with VEGF (50 ng/mL) for 5 minutes. Cell lysates were subjected to phospho-Akt1–Ser473- or total Akt1-ELISA analysis. (B) After 48 hours, HUVECs were serum-starved for 3 hours and then left untreated or stimulated with VEGF (50 ng/mL) for 10 minutes. Cell lysates were subjected to Western blot analysis using phospho Erk1/2 or total Erk1/2. (C) Transfected HUVECs were lysed 48 hours after transfection. Cell lysates were subjected to Western blot analysis using antibodies against phospho-Y397-FAK, FAK, Rap1, and tubulin.