Fig. 4

Crosstalk between mTORC1 and mTORC2 during NK cell activation. a Immunoblotting to detect the phosphorylation (p-) of S6 at ser235/236 (p-S6^ser235/236) and Akt at Ser473 (p-Akt^ser473) in NKL cells after stimulation with IL-15 (10 ng/ml) (left) or IL-15 (100 ng/ml) (right) for the indicated time periods. β-Actin was used as an internal control. The data shown are representative of three independent experiments. Uncropped gels are shown in Supplementary Fig. 8. b Intracellular flow cytometric analysis of p-S6^ser235/236 and p-Akt^ser473 in murine splenic NK cells (CD3⁻CD19⁻NK1.1⁺) after stimulation with IL-15 (100 or 10 ng/ml) for the indicated time periods. The MFI of p-S6 ^ser235/236 or p-Akt^ser473 at each time point was normalized to the baseline reading at the 0-h time point for each individual mouse. Four independent individual mice were used. c–e Flow cytometric analysis and cumulative results for p-Akt^Ser473 (c), CD122 (d), and p-STAT5^Y694 (e) in splenic NK cells (CD3⁻CD19⁻NK1.1⁺) from control versus Raptor^∆NK mice. f Intracellular flow cytometric analysis and cumulative results for p-S6 ^ser235/236 in total splenic NK cells (CD3⁻CD19⁻NK1.1⁺) and the indicated subpopulations thereof from control versus Rictor^∆/∆ (top) or control versus Rictor^∆NK (bottom) mice. g Statistical quantification of p-S6^ser235/236 (MFI relative to the untreated control littermates) in splenic NK cells (CD3⁻CD19⁻NK1.1⁺) with or without IL-15 stimulation for 4 h ex vivo. The dashed line indicates the control group, and the solid line indicates the gene knockout group. The MFI was calculated relative to total NK cells or to the CD27SP subset of NK cells from control littermates. CD27SP, DP, and CD11bSP represent CD27⁺CD11b⁻, CD27⁺CD11b⁺, and CD27⁻CD11b⁺ NK cell subsets, respectively. Each dot represents one mouse, and all the experiments were replicated 3 (c–e, f bottom, g), or 7 (f top) times; error bars represent SD; ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001; one-way ANOVA (b, g); unpaired two-tailed Student’s t test with Welsh’s correction (c–f)