c-Kit⁻ [Lin Sca-1 IL7Rα]^−/low cells from CMML mice show hyperactivation of the ERK and Stat5 pathways. Total bone marrow cells isolated from CMML mice or control mice were serum- and cytokine-starved for 1 hour and stimulated with various concentrations of GM-CSF (0, 0.16, 0.32, 2, and 10 ng/mL) at 37°C for 10 minutes. Levels of phosphorylated ERK1/2 were measured using phospho-specific flow cytometry. Nonneutrophil bone marrow cells were gated for data analysis. Representative gating strategy and plots of p-ERK1/2 (A) and p-Stat5 (B) are shown. Myeloid progenitors are enriched in c-Kit⁺ [Lin Sca-1 IL7Rα]^−/low cells as described in Figure 4 (G1 cells), whereas G2 cells are defined as c-Kit⁻ [Lin Sca-1 IL7Rα]^−/low cells. To quantify the activation of ERK1/2 and Stat5 in CMML cells and control cells, median intensities of p-ERK1/2 and p-Stat5 at different GM-CSF concentrations are compared with their respective control cells at 0 ng/mL, which is arbitrarily set at 1. Representative results of 3 experiments of more than 10 independent experiments are shown.