The 4E-BP1 T^37/46 priming residues are mTOR-dependent and limiting for translation initiation. (A) Primary AML cells and the MV4-11 cell line were cultured for 1 hour without or with 100 or 1000 nmol/L Ku-0063794. (B) Primary AML cells were transfected with mTOR, raptor, or rictor siRNAs. (C) HEK293 cells were transfected with 4E-BP1^WT, 4E-BP1^A37A46, 4E-BP1^E37E46, and Pim-2 plasmids and then, eventually, treated for 24 hours with 10mM metformin, as indicated. At 48 hours after transfection, cells were lysed in 10% NP-40 lysis buffer and immunoprecipitations were performed using anti-HA antibodies. Immunoprecipitated proteins and whole-cell lysates were then subjected to Western blotting.