T cell–specific deletion of Rac1/Rac2 inhibits TCR-induced cell growth and signaling. Single-cell suspensions of thymocytes or splenic T cells were prepared from mice of the indicated genotypes. The cells were plated on 96-well plates at 2 × 10⁵/well in 100 μL culture with or without anti-TCR antibody or IL-2 (A-C). The medium was collected at day 2 from the thymocyte culture for IL-2 assay by ELISA (B), and the cell numbers were counted after a 3-day culture (A,C). Data are expressed as the fold of growth relative to the number of WT cells without stimulation. Error bars represent the standard deviations of 5 WT and 5 Rac1^−/−Rac2^−/− mice. *P < .05; **P < .01. (D) Western blotting was performed to assess the phosphorylation status of Akt, Erk, p38, and ZAP70 after a 3-minute TCR cross-linking of freshly isolated thymocytes. The quantitative results were shown as ratios between normalized phospho-Akt, -Erk, -p38, or -ZAP70 and total Akt, Erk, p38, or ZAP70, and are presented as means plus or minus SD from 5 WT and 5 Rac1^−/−Rac2^−/− mice. Rac1 expression was probed by anti-Rac1 blotting in parallel. Error bars represent SD.