DEPTOR inhibits ERK1/2 and STAT1 activity in ECs. HUVECs were transfected with control or DEPTOR siRNAs (#1 and #2), and after 48 hours, a phosphokinase protein array was performed on cell lysates to analyze the relative expression of 46 individual phosphokinase proteins. (A) A representative blot (of n = 2) showing the levels of phosphorylation of individual kinases and their protein substrates in control and 2 DEPTOR siRNA-transfected ECs (#1 and #2). Transfection with either siRNA resulted in similar findings by phosphokinase array. (B) Expression of pS6K1 (T389), pAkt (S473), pERK1/2 (T202/Y204), pSTAT1 (Y701), and GAPDH was examined in control or DEPTOR siRNAs-transfected HUVECs by western blot analysis. Representative results of >3 independent experiments are shown. (C-D) HUVECs were transfected with control or DEPTOR siRNAs, cultured for 48 hours, and treated with (C) rapamycin (10 ng/mL) or (D) Torin1 (1 μM) and/or U0126 (10 μM) for the last 18 hours of cell culture. Cells lysates were analyzed by western blot analysis for the expression of pS6K1 (T389), total S6K1, pAkt (S473), total Akt, pERK1/2 (T202/Y204), total ERK1/2, pSTAT1 (Y201 and S727), total STAT1, and GAPDH, as illustrated. Representative blots from 3 independent experiments are shown.