STAT5 regulation of Akt activation requires STAT5-activated transcription. (A) Cells were transfected with control or STAT5a-1*6 expression plasmids and cultured overnight. Cells were then washed and cultured with or without IL-3 for an additional 8 hours and cell lysates were immunoblotted for phospho-STAT5, phospho-Akt, and Actin. (B,C) IL-7Rα–expressing FL5.12 cells that expressed Bcl-xL to maintain cell viability were transfected with control (GFP) or STAT5a/b shRNAi and cultured for 30 hours, washed, and cultured in the absence or presence of IL-7 for an additional 14 hours. (B) STAT5, phospho-Akt, and Actin were observed by immunoblot. (C) Phospho-Akt/Actin ratios were quantitated from untreated and IL-7–treated samples from 4 independent experiments to determine IL-7–stimulated phospho-Akt in control and STAT5a/b shRNAi treated cells. (D) IL-7Rα–expressing FL5.12 cells were washed and cultured without cytokine for 6 hours, treated with DMSO (vehicle), LY294002, or actinomycin D for 30 minutes, and stimulated with IL-7 for 8 hours. (E) T cells were treated with DMSO (vehicle), LY294002, or actinomycin D for 30 minutes, and stimulated with IL-7 for 8 hours. (D,E) Cell lysates were analyzed by immunoblot for phospho-STAT5 (Y659), STAT5, Pim1, phospho-Akt (S473), Akt1, phospho-FoxO1/FoxO3a (T24/T32), and Actin. (F) Cells were transfected with control (C), STAT5a-1*6 (5*), or STAT5a-1*6-VVV (5*V) expression plasmids and cultured overnight. Cells were then washed and cultured in the absence of cytokine for 9 hours. Cell lysates were analyzed by immunoblot for phospho-STAT5 (Y694), STAT5, Pim1, phospho-p85 (Y458), p85α, phospho-Akt (T308), Akt1, Akt2, PTEN phospho-mTOR (S2448), and Actin. Values represent means plus or minus SEM from 4 independent experiments. By Student t test, *P < .05. White vertical lines have been inserted to indicate repositioned gel lanes in panel A.