IFNα-dependent activation of Stat complexes in KT-1 cells is mTOR independent. (A) KT-1 cells were preincubated with rapamycin for 60 minutes and were subsequently treated with IFNα, as indicated. Top panel shows an immunoblot with an anti-phospho-Tyr701 Stat1 antibody. Bottom panel shows reprobing the same blot with an anti-Stat1 antibody. (B) Similar experiment as in panel A, except that an antibody against the phosphorylated form of Stat1 on serine 727 was used in the immunoblot shown in the top panel. (C) Actively growing KT-1 cells were preincubated for 30 minutes with rapamycin, as indicated. The cells were subsequently treated with IFNα, as indicated. Nuclear extracts were reacted with 40 000 cpm of a ³²P-labeled interferon-stimulated response element (ISRE) probe and complexes were resolved by native gel electrophoresis and visualized by autoradiography. (D) Similar experiment to the one shown in panel C, except that a ³²P-labeled SIE probe was used.