Unaffected thymocyte development and positive selection in mice lacking H-ras or N-ras. (A) Cells from WT, H-ras–, and N-ras–deficient mice were stained for expression of CD4, CD8, and CD3 and were analyzed by flow cytometry. Numbers within dot plots indicate percentage of cells in each quadrant. Bottom panels show CD3 expression in thymocytes (left) and splenic CD4⁺ T cells (right) from WT (shaded curve), H-ras KO (solid line), and N-ras KO (dotted line) mice. (B) CD4 and CD8 expression in AND TCR transgenic thymocytes (gated as TCR^hi) from Ras-sufficient (+/+) and deficient (−/−) littermate mice. Numbers indicate percentage of cells in the gated region. Clonotypic TCR expression in gated CD4⁺ SP thymocytes from Ras-sufficient (solid line) and deficient (dotted line) mice is shown in the bottom panels. (C) CD4⁺ SP thymocyte numbers in Ras-sufficient and -deficient and TCR transgenic mice. Results from individual mice (open circles) and the average for each population (thick bars) are shown. (D) Thymocytes were stimulated with the indicated concentration of moth cytochrome c (MCC) 81-103 peptide for 48 hours and stained for expression of CD4, CD8, and CD69. Mean CD69 expression ± SD (n = 2) in gated CD4⁺ SP cells is shown. (E) CD4, CD8, and clonotypic TCR expression in thymocytes from Ras-sufficient and -deficient OT-I TCR transgenic mice. CD8⁺ SP frequencies were (n = 4): H-ras (+/+: 13 ± 3.4; −/−: 13 ± 2.0; P = .98); N-ras (+/+: 8.7 ± 5.9; −/−: 8.9 ± 6.0; P = .96). (F) ERK activation in anti–CD3-stimulated thymocytes. Fold induction of ERK relative to unstimulated (time 0) cells is indicated. Total AKT is shown as loading control.