Figure 1.

Sos1 loss abolishes activation of WT Nras and Hras and decreases GM-CSF signaling in Kras^G12D/+cells. Control (C), Sos1^fl/fl;Vav-Cre (Sos1^−/−), Kras^{LSL G12D/+};Vav-Cre (Kras), Kras^{LSL G12D/+};Sos1^fl/fl;Vav-Cre (Kras; Sos1^−/−), Nras^{LSL Q61R/+};Vav-Cre (Nras), and Nras^{LSL Q61R/+};Sos1^fl/fl;Vav-Cre (Nras; Sos1^−/−) mice were sacrificed at age 6 weeks. (A-B) Sos1 expression levels in bone marrow (BM) cells of control, Kras (A), and Nras (B) mice were quantified against the levels of β-actin, using ImageStudioLite software. The ratios in control cells are arbitrarily set at 1. ns, not significant. (C) Whole-cell lysates were extracted from BM cells and immunoprecipitated with anti-Ras G12D antibody or preimmune immunoglobulin G (IgG). The resulting precipitates were immunoblotted with anti-Sos1 or anti-Ras antibody. (D) Whole-cell lysates were extracted from BM cells 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 whole-cell lysates, using a 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. Oncogenic Kras was detected using an antibody specifically against Ras G12D protein and quantified against the levels of β-actin. (E) BM cells were serum- and cytokine-starved for 2 hours at 37°C. Cells were then stimulated with 2 ng/mL mGM-CSF for 10 minutes at 37°C. Levels of p-ERK1/2 and p-STAT5 were measured using phosphoflow cytometry. Myeloid progenitors (MP) are enriched in Lin^−/low c-Kit⁺ cells and myeloid precursors (MPre) are enriched in Lin^−/low c-Kit⁻ cells. Data are presented as mean + standard deviation. *P < .05; **P < .01; ***P < .001.