N-Ras^G12D,C181S mislocalization is associated with normal cytokine responses despite elevated Ras-GTP levels. (A) Levels of total Ras and Ras^G12D protein in BM whole-cell lysates (WCL), cytoplasmic fraction (Cyto.), and solubilized membrane fraction (Mem.) from a representative western blot (n = 3). Na, K-ATPase, and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) were used as membrane and cytoplasm markers, respectively. (B) Raf binding domain (RBD) pulldown assays (right lanes) were performed to measure Ras-GTP levels in starved (−) or GM-CSF–stimulated (+) Mac-1⁺ BM cells (n = 3). Heat shock protein 90 was used as loading control. (C) Phospho-flow cytometric analysis of pERK and pS6 in Mac-1⁻ Gr-1⁻ c-Kit⁺ cells stimulated for 10 minutes with increasing doses of SCF showing representative plots (left) and median fluorescence intensity (MFI) of pERK (top right) and pS6 (bottom right) normalized to starved cells from WT mice. Bar graphs indicate mean ± SEM (n = 4). (D) Proliferation of Mac-1⁻ c-Kit⁺ cells in response to increasing doses of GM-CSF. Mean ± SEM from 3 biologic replicates. Med., cytokine-free medium. *P < .05; **P < .01; ***P < .001.