Biochemical and functional analysis of WT and

Nf1^+/− thymocytes. (A) Ras activity of freshly isolated WT (lane 1) and Nf1^+/−(lane 2) thymocytes. GTP-bound Ras levels were determined by incubating cell lysates with Raf-1 Ras binding domain (RBD) conjugated to agarose beads and by immunoblotting with an anti-Ras antibody. Immunoblots and quantitative densitometry for Ras-GTP levels and Western blots of total Ras are shown. (B) Thymic cellularity of WT andNf1^+/− mice. Single cell suspensions from WT and Nf1^+/− thymi were prepared and counted. Bars represent the absolute numbers of thymocytes (± SEM), n = 12 and * P < .0004. (C) T-cell subset distribution of WT and Nf1^+/− thymi. Bars represent the absolute numbers of indicated T-cell subsets (± SEM), n = 12 and * P < .004. (D) Immunocytometric analysis of thymocytes from WT and Nf1^+/− mice. Thymocytes from WT andNf1^+/− mice were stained with fluorochrome-conjugated antibodies directed against the indicated cell surface proteins and analyzed by flow cytometry. Data are representative of 5 independent experiments. (E) Altered proliferation of Nf1^+/− thymocytes. Thymocytes (10⁵/well) were isolated and activated with plate-bound anti-CD3 mAb, IL-2, or anti-CD3 mAb plus IL-2. Proliferation was measured by a thymidine incorporation assay. Bars denote the mean thymidine incorporation (cpm ± SEM) of a representative experiment performed in replicates of 6. * P < .01Nf1^+/− versus WT. ** P < .01Nf1^+/− versus WT.