Figure 4. Rictor-deficiency caused suboptimal TCR signaling.

A. Rictor^T+/+ and rictor^T−/− DP thymocytes were stimulated in vitro for the indicated times with 10 µg/mL CD3ε mAb. Phosphorylation of ERK (T202/Y204) as well as the HM of Akt (S473) was analyzed by immunoblotting with the specific phospho-antibodies and quantitated by densitometry. Membranes were stripped and reblotted with sera against the native form of the respective kinases. Shown is a representative experiment out of three as well as a quantitative plot of fold increase of protein phosphorylation as compared to the non-stimulated kinases from wild-type cells (n= 3). B. Thymocytes were stimulated for 10 min with PMA (16 nM) and lysates were subjected to immunoblotting. Shown are representative blots of three experiments. C. Thymocytes of 3 rictor^T+/+ and 3 rictor^T−/− littermates were harvested, counted and lysates were resolved by immunoblotting. Shown are representative blots of three experiments. D. Thymocytes were cultured, ex vivo at 37 ° C for 1 to 4 days. Following cell staining with Annexin V and propidium iodide (PI), viability was measured at various time points by flow cytometric analysis (n=4).