TCR- and DAG-induced mTORC1 and mTORC2 activation. WT thymocytes (A-B) and splenic T cells (C-F) were left unstimulated or stimulated with an anti-CD3 antibody (500A2; A-D) at 37°C for the indicated times (in minutes) or with PMA (E-F) 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 at least 3 experiments. Panels B, D, and F show quantification for phosphorylation levels relative to unstimulated controls (means ± SEM of 3 independent experiments; *P < .05; **P < .01; ***P < .001 relative to unstimulated control as indicated by ANOVA).