Inhibition of TCR-induced mTOR activation by DGKα and DGKζ. (A) Establishment of DGKζ1-, KD-DGKζ1-, or DGKζ2-expressing 2B4 T-cell lines by retroviral infection as described in Figure 4A. (B-C) Enhanced DGK activity inhibits TCR-induced mTOR activation. 2B4 cells overexpressing DGKζ1, KD-DGKζ1, or DGKζ2 were left unstimulated or stimulated with anti-CD3 (500A2) 5 minutes at 37°C. Cell lysates were separated by SDS-PAGE followed by immunoblotting with the indicated antibodies. The blots were stripped and reprobed with an anti–β-actin antibody for a loading control. Data shown are representative of/quantified from 4 experiments. (D-E) Enhanced TCR-induced mTOR activation in DGKα and DGKζ double-deficient thymocytes. WT and DGKαζDKO (DKO) thymocytes were not pretreated or pretreated with U0126 (U0) or LY294002 (LY) at 37°C for 30 minutes, and were then left unstimulated or stimulated an anti-CD3 antibody (500A2) at 37°C for 5 minutes. Cell lysates were separated by SDS-PAGE followed by immunoblotting with the indicated antibodies. The blots were stripped and reprobed with an anti–β-actin antibody for a loading control. Data shown are representative of/quantified from 5 experiments. (F) Schematic illustration of mTOR activation in T cells (please see “Discussion” for details).