Pim-2 is constantly expressed in AML and controls 4E-BP1 S⁶⁵ phosphorylation and subsequent cap-dependent translation in AML. (A) Samples from AML patients or normal CD34⁺ hematopoietic cells from healthy donors were starved for 4 hours in cytokine- and serum-free medium. A total of 10⁶ cells were lysed in boiling Laemmli sample buffer and Western blot for Pim-2 was performed, with 50 ng of human recombinant Pim-2 (rh Pim-2, from Cell Signaling Technology) as control for Pim-2 detection. Actin was used as the loading control. (B) Pim-2 siRNA or nontargeted control siRNA were transfected into AML blast cells, with the Amaxa nucleofector system. At 24 hours after electroporation, cell extracts were analyzed by Western blot to assess the expression of Pim-2, c-Myc, Cyclin D1, P70S6K, 4E-BP1, BAD, and actin and the phosphorylation level of P70S6K on T³⁸⁹, BAD on S¹¹⁸, and of 4E-BP1 on T^37/46 and S⁶⁵ residues. The signal intensity was quantified using the Multi Gauge, Version 3.0 software from Fuji. Each histogram represents the mean of 4 independent experiments, and results are expressed as a ratio between the Pim-2, c-Myc, Cyclin D1, or the phospho-4E-BP1 S⁶⁵ signals and the actin signal, relative to the control condition. Vertical bars represent SD.