mTORC1 regulates glucose uptake and glycolysis in IL-2–maintained CTLs. (A) Immunoblot analysis for Glut1 expression in naive OTI CD8⁺ T cells ± TCR (SIINFEKL) stimulation for 20 h and also mature OTI CTLs. The black line indicates that intervening lanes have been spliced out. (B) Immunoblot analysis for Glut1 expression in CTLs treated with or without IL-2 for 20 h. 4EBP1 was used as a loading control. (C and D) Analysis of glucose uptake (C) and lactate production (D) in P14-LCMV CTLs treated with or without IL-2 for 20 h. (E and F) Analysis of lactate production (E) and glucose uptake (F) in P14-LCMV CTLs treated with or without rapamycin for 20 h. (G) Immunoblot analysis for Glut1 expression in P14-LCMV CTLs treated with or without rapamycin for 20 h. 4EBP1 was used as a loading control. (A–G) Data are mean ± SEM or representative of at least three experiments. All metabolic assays were preformed in triplicate (**, P < 0.01; ***, P < 0.001). Molecular mass is indicated in kilodaltons. (H) SILAC-based proteomic analysis of P14-LCMV CTLs treated with and without rapamycin for 48 h. Shown is the relative expression of rate-limiting glycolytic enzymes (rapamycin/untreated). Data are mean ± SEM for three experiments and were analyzed by ANOVA (**, P < 0.01; ***, P < 0.001). CD8α was used as a control protein with unchanged expression.