Activation of p70 S6 kinase by IFNα in BCR-ABL-expressing cells is PI 3′ kinase- and mTOR-dependent. (A) KT-1 cells were treated with IFNα for the indicated times. Top panel shows an immunoblot with an antibody against phosphorylated mTOR on serine 2448. Bottom panel shows reprobing of the same blot with an anti-mTOR antibody. (B) KT-1 cells were pretreated for 30 minutes with either LY294 002 (50 μM) or rapamycin (20 nM) prior to treatment with IFNα for 30 minutes as indicated. Top panel shows an anti-phospho-p70 S6 kinase immunoblot. Bottom panel shows reprobing of the same blot with an anti-p70 S6 kinase antibody. (C) KT-1 cells were pretreated for 30 minutes with the various pharmacologic inhibitors prior to treatment with IFNα for 30 minutes, as indicated. Cell lysates were immunoprecipitated with an anti-p70 S6 kinase antibody or control nonimmune rabbit immunoglobulin (RIgG). In vitro kinase assays to detect p70 S6 kinase activity were then carried out on the immunoprecipitates. The data represent means plus or minus the standard error (SE) of 3 independent experiments.