DEPTOR associates with mTORC1 and inhibits its activity in ECs, but it minimally interacts with mTORC2. (A) DEPTOR, mTOR, Raptor, and Rictor were immunoprecipitated (IP) from HUVECs and HEK293 cells, and western blot (WB) analysis was subsequently performed with anti-DEPTOR, -mTOR, -Raptor, and -Rictor. As illustrated, DEPTOR associates with mTOR and raptor, but minimally associates with Rictor in HUVECs. (B) HUVECs were transfected with a control or 2 DEPTOR siRNAs (#1 and #2), and the expression of pS6K1 (T389), S6K1, pAkt (S473), and Akt was evaluated by western blot analysis. (C) Control or DEPTOR siRNA (50 nM)-transfected ECs were cultured for 48 hours and treated with rapamycin (10 ng/mL) for the last 1 hour of culture. Cell lysates were analyzed by western blot for the expression of pS6K1 (T389), S6K1, pAkt (S473), and Akt. (D) The expression of pSGK (S422), SGK, pS6K1 (T389), S6K1, pAkt (S473), and Akt was analyzed by western blot in HUVECs transfected with a control or 2 DEPTOR siRNAs (50 nM). All data presented are representative of ≥3 independent experiments.