IL-7 leads to rapid STAT5 activation yet delayed and sustained Akt activation, both of which promote glucose uptake. (A) Control (C), IL-7Rα (7R)–, and IL-7Rα-Y449F (449)–expressing FL5.12 cells were washed and cultured without cytokine (t = 0) and then stimulated with IL-3 or IL-7 for 15 minutes or 6 hours. Cell lysates were analyzed by immunoblot for phospho-STAT5 (Y694), Pim1, phospho-Akt (S473), Akt1, Akt2, and Actin. (B, C) IL-7Rα cells were washed and cultured without cytokine for 6 hours, then stimulated with IL-7 for 15 minutes to 24 hours. Cell lysates were analyzed by immunoblot for (B) phospho-Akt (S473) and Akt1 and (C) phospho-STAT5 (Y694), STAT5, Pim1, phospho-p85 (Y458), p85α, phospho-Akt (T308), Akt1, Akt2, phosho-mTOR (S2448), and Actin. (D) Purified primary T cells were analyzed without stimulation or after culture for 15 minutes or 18 hours with IL-7 or IL-4. Cell lysates were analyzed by immunoblot for phospho-STAT5 (Y694), total STAT5, phospho-Akt (S473), Akt1, and Actin. (E,F) FL5.12 cells were transfected with Bcl-xL as a control, MyrAkt1, or STAT5a-1*6 expression plasmids, cultured with or without IL-3 for 10 hours, and analyzed for (E) phospho-Akt and (F) phospho-STAT5 and Actin. Note that short exposures are shown to illustrate levels of phosphorylation in constitutively active proteins. (G) One day after transfection, Bcl-xL, MyrAkt1, or STAT5a-1*6 transfected cells were cultured in the absence of cytokine for 14 hours and glucose uptake was measured. Values represent means plus or minus SEM of triplicate samples within the given experiment. By Student t test, *P < .005, **P < .001. White vertical lines have been inserted to indicate repositioned gel lanes in panels D and F.