(A) Activated and expanded human T cells were resuspended in fresh RPMI (10% FBS) devoid of IL-2. AKT phosphorylation was assessed before (B) and after (0.5, 4, and 24 hours) cytokine withdrawal by Western blot. A representative blot is shown (n = 5). (B) Cumulative data of 5 experiments. Mean ± SEM of pAKT:AKT density is shown. **P ≤ 0.01 (paired 2-tailed t test). (C) Human T cells activated and expanded were lysed at the indicated time points. Phosphorylation of the enlisted proteins was analyzed by immunoblot (n = 2). (D) T cells from WT and cKO mice were activated and expanded. Cells were lysed at the indicated time points after IL-2 deprivation. AKT phosphorylation was quantified by Western blot. Densitometry is indicated under each blot. Two-way ANOVA with Bonferroni’s multiple comparisons test; *P = 0.025, ***P = 0.0006 (n = 4). (F) Human T cells were infected with B55β-specific or control shRNA-encoding lentiviruses. Cells were deprived of IL-2, and pAKT S473 was quantified by flow cytometry. Representative histograms: gray shades indicate pAKT prior and empty histograms pAKT after 8 hours of cytokine withdrawal. (G) Mean ± SEM of pAKT (S473) in cells infected with control (blue circles) or B55β-specific (orange circles) lentiviruses. Mean fluorescence intensity (MFI) was normalized to basal time point. Paired 2-tailed t test; **P ≤ 0.01. (H) The percentage of pAKT⁺ cells from F was quantified before (empty circles) and after IL-2 withdrawal (full circles). Each dot represents one sample. Two-tailed t test, **P = 0.001. (I) 293 T cells were transfected with a B55β-encoding plasmid (0.5–10 μg). Twenty-four hours later, cells were lysed and the indicated proteins were quantified by Western blotting. Densitometry is indicated. Cumulative data of 4 independent experiments showing AKT:β-actin (J) and pAKT:AKT (K) are shown.