Somatic expression of oncogenic Nras at its endogenous level does not lead to acute development of myeloproliferative disease. Six-week-old control and Nras G12D mice were injected with pI-pC as described in “Mice.” One week after last injection, different tissues were isolated and analyzed. (A) Schematic illustration of floxed and activated oncogenic Nras alleles. (B) Genotyping analysis of genomic DNA to detect wild-type allele, LSL allele, and recombined LSL allele (1 LoxP allele). (C) Total RNA was extracted from bone marrow cells. Direct sequencing of RT-PCR–amplified Nras gene using a reverse primer to confirm the sequences at the codon 12 (underlined in red). Arrows indicate the wild-type and mutated nucleotide at the codon 12. (D) Levels of Nras-GTP, the active form of Nras, were analyzed in lysates extracted from bone marrow cells by affinity purification (AP) of lysates using a glutathione S-transferase fusion with the Ras binding domain of Raf (Raf RBD) immobilized on agarose beads followed by Western blot analysis using an antibody against Nras. The top panel illustrates the total input levels of Nras proteins. (E) Peripheral blood samples were collected from Nras G12D mice and control mice. Debris and unlysed red blood cells (low forward scatter) and dead cells (propidium iodide positive) were excluded from analysis. The percentages of T cells (Thy1.2), B cells (CD19), and myeloid cells (Mac1 and Gr1) are indicated on each plot.