mTORC2-dependent Akt signaling inhibits Raf1-MEK1/2-ERK1/2, leading to defective induction of IRF-1. (A) Western blot analysis of ECs treated with rapamycin for the indicated times. (B, top) Western blot analysis of ECs treated with the indicated doses of AktVIII for 24 h. (bottom) Western blot analysis of ECs treated with 4 µM AktVIII for 24 h and then stimulated with TNF for the indicated times. (C) ECs were pretreated with the indicated doses of AktVIII and then stimulated with TNF overnight. VCAM-1 expression was assessed by FACS. (D) ECs transduced with the indicated constructs were pretreated with rapamycin, stimulated with TNF for the indicated times, and assayed by Western blotting. (E) ECs transduced with the indicated constructs were pretreated with rapamycin and stimulated with TNF and then assayed by FACS. Data are normalized to MFI of VCAM-1 staining in empty vector–ECs treated with TNF. (F) ECs transduced with the indicated constructs were stimulated with TNF for the indicated times and assayed by Western blotting. (G) ECs expressing myristoylated Akt were transduced with control or Raf 22W vector. Transduced ECs were pretreated with rapamycin or U0126 and stimulated overnight with TNF. VCAM-1 expression was assessed by FACS. (H) Western blot analysis of control- or rapa-ECs stimulated with TNF for the indicated times. (I) mRNA expression in control- and rapa-ECs stimulated with TNF for 0.25 h. Veh, vehicle. (J) Western blot analysis of ECs pretreated with rapamycin and/or U0126 and then stimulated with TNF for 2 h. (K) ECs transduced with the indicated constructs were pretreated with rapamycin, stimulated with TNF for 2 h, and assayed by Western blotting. (L) ECs were transduced with control or IRF-1 shRNA vectors, as indicated. Transduced ECs were then pretreated with rapamycin and stimulated overnight with TNF, as indicated. VCAM-1 expression was assessed by FACS after TNF stimulation. (M) ChIP analysis of control and rapa-ECs stimulated with TNF for 2 h. DNA quantification is expressed as percent input and normalized to TNF treatment sample. ND, not detected. For A, B, D, F, H, J, and K, similar results were seen in two independent experiments. Mean ± SEM are shown for data pooled from two (C [n = 6], E [n = 6], G [n = 6], I [n = 3], L [n = 6], and M [n = 4]) independent experiments (n = total replicates). For E, G, and L, significance was determined by ANOVA with Tukey’s post-hoc test; all other data were analyzed with the Student’s t test. *, P < 0.05.