Kras^−/−; Nras^Q61R/+ thymic T cells show hyperactivation of Nras/MEK/ERK signaling and hyperproliferation. Six- to 7-week-old Mx1-cre (Con), Kras^−/− (K−/−), Nras^Q61R/+ (Q/+), and Kras^−/−; Nras^Q61R/+ (K−/−; Q/+) mice were treated with pI-pC as described in "Materials and methods." Thymocytes were collected for analysis on day 37. (A) Whole cell lysates (WCL) were extracted and analyzed for expression levels of different Ras isoforms, which were quantified against the levels of β-actin using ImageStudioLite software. Ras-GTP was affinity purified from WCL using a glutathione S-transferase (GST) fusion with the Ras binding domain of Raf (Raf RBD) immobilized on agarose beads. The levels of Ras-GTP bound forms were quantified against the levels of their corresponding Ras isoforms. The ratios of Ras-GTP/Ras in control cells are arbitrarily set at 1. (B) Phosphorylated ERK1/2 (pERK1/2) in the indicated populations of thymocytes were quantified with phospho-flow assay. Representative results from 1 set of experiment and quantification of 6 to 9 independent experiments were shown. (C) Cell-cycle analysis of different populations of thymocytes using 4′,6-diamidino-2-phenylindole (DAPI) staining. Data are presented as mean – SD. P values were calculated with a 2-tailed Student t test. *P < .05; **P < .01.