Overexpression of oncogenic Nras leads to hyperactivation of ERK1/2 signaling in AML MPs. (A) Representative sequencing results from RNA-Seq analysis show UPD of Nras^G12D mutation in AML MPs. (B) Genotyping analysis of the Nras locus in multiple AML samples isolated from independent recipients (spleen weight, >1 g). (C) RPKM of Nras in control and AML MPs. (D) Western blot analysis of Nras expression levels in AML and wt spleens. (E) Whole bone marrow cells isolated from control, p53^−/−, Nras^G12D/+, and Nras^G12D/+p53^−/− mice on day 12 and moribund AML Nras^G12D/+p53^−/− recipients were serum and cytokine starved for 2 hours and stimulated with various concentrations of murine GM-CSF (0, 0.32, and 10 ng/mL) at 37°C for 10 minutes. Levels of phosphorylated ERK1/2 (p-ERK1/2) were measured using phosphor-specific flow cytometry. Non-neutrophil bone marrow cells were gated for data analysis. Myeloid progenitors are enriched in Lin^–/lowc-Kit⁺ cells, whereas myeloid precursors are enriched in Lin^–/lowc-Kit^– cells. To quantify the activation of ERK1/2, median intensities of p-ERK1/2 at different GM-CSF concentrations are compared with their respective control cells at 0 ng/mL, which is arbitrarily set at 1. Data are presented as mean ± SD. *P < .05; **P < .01; ***P < .001.