IFNα-dependent phosphorylation of 4E-BP1 repressor of mRNA translation in KT-1 cells. (A) KT-1 cells were treated with IFNα for different times, as indicated. Top panel shows an immunoblot with an antibody against phosphorylated 4E-BP1 on Thr 37/46. Bottom panel shows reprobing the same blot with an anti-4E-BP1 antibody. (B) Similar experiment to the one shown in panel B, except that immunoblotting was first performed using an antibody against the phosphorylated form of 4E-BP1 on Thr 70. (C) Similar experiment to the one shown in panel B, except that the cells were incubated for longer times with IFNα, as indicated. (D) KT-1 cells were preincubated with LY294 002 (50 μM) or rapamycin (20 nM) for 30 minutes and were subsequently treated with IFNα, as indicated. Top panel shows an immunoblot with an antibody against phosphorylated 4E-BP1 on Thr 37/46. Bottom panel shows reprobing of the same blot with an anti-4E-BP1 antibody. (E) KT-1 cells were pretreated with LY294 002 (50 μM) or rapamycin (20 nM) for 30 minutes and were subsequently incubated with IFNα, as indicated. Top panel shows an immunoblot with an antibody against phosphorylated 4E-BP1 on Thr 70. Bottom panel shows reprobing of the same blot with an anti-4E-BP1 antibody. (F) Similar experiment to the one shown in panel E, except that IFNβ was used instead of IFNα.