Pim-2 mediates 4EBP-1–independent cell growth and survival. (A) Cell size 24 hours after transfection and growth factor withdrawal shown for FL5.12 cells expressing Bcl-X_L, myristolated Akt-1 (mAkt), or Pim-2 transiently transfected with empty vector (Vector) or an HA-tagged 4EBP2A expression vector in which threonines 37/46 have been mutated to alanines (4EBP2A). Measurements shown are forward scatter of live cells from each sample from triplicate samples and indicate the mean ± SD. (B, left) Cell viability measurements after 48 hours of IL-3 withdrawal are shown for FL5.12 cells described in panel A. Data shown are the mean ± SD of 3 independent samples. (Right) Lysates prepared from these cells were probed for the expression of Bcl-X_L, phospho-serine 473 Akt (p-473 Akt), Pim-2, HA-4EBP2A, and Actin. (C) Lysates generated from FL5.12 cells expressing Bcl-X_L, myristolated Akt (mAkt), or Pim-2 grown in the presence (+IL-3) or absence (-IL-3) of IL-3 for 24 hours were probed for the expression of phospho-serine 209 eIF4E (p-209 eIF4E), total eIF4E, and Actin. (D) Viability is shown 24 hours after IL-3 withdrawal for Pim-2–expressing FL5.12 cells stably transfected with a plasmid expressing an shRNA targeting eIF4E and control. (Inset) Lysates were probed for the expression of eIF4E, Pim-2, and Actin. (E) Lysates were generated from IL-3–dependent bone marrow cell lines described in Figure 3. The genotype of each cell line is indicated above the immunoblots. Western blots of lysates were probed for the expression of phospho-threonine 37/46 4EBP-1, 4EBP-1, Actin, phospho-serine 209 eIF4E, eIF4E, and Actin as indicated.