Mek activity modulates TCR-induced mTOR activation. (A) Establishment of caMek1- and dnMek1-expressing 2B4 T-cell lines. 2B4 T cells were infected with retrovirus expressing caMek1 and dnMek1, respectively. Mek1/2 expression in infected cells after sorting for positive GFP was determined by Western blot with an anti-Mek1/2 antibody. The blot was also probed with an anti–β-actin antibody for a loading control. (B-C) caMek1- or dnMek1-expressing 2B4 T cells were either not pretreated or pretreated with rapamycin (Rapa) or LY294002 (LY) for 30 minutes at 37°C, and then left unstimulated or stimulated with 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 2 experiments. (D-G) Effects of Mek1/2 and PI3K inhibition on TCR-induced mTOR activation. WT thymocytes (D-E) and splenic T cells (F-G) were not pretreated or pretreated with U0126 (U0) or LY294002 (LY) at 37°C for 30 minutes, and were then left unstimulated or stimulated with 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. (H-I). Effects of PI3K and Mek1/2 inhibitors on PMA-induced mTORC1 activation. WT thymocytes were pretreated with U0 or LY for 30 minutes, followed by stimulation with PMA for 5 minutes. Data shown are representative of/quantified from 3 experiments.