A C181S mutation prevents MPN in homozygous Nras^G12Dmice. (A) BM complementary DNA sequencing indicating the positions of the G12D (left) and C181S (right) mutations (n = 3). (B) Nras transcript levels in BM cells (n = 3). (C-D) Representative western blot showing protein levels of total Ras (C) and Ras^G12D proteins (D) in BM cells (n = 5). Heat shock protein 90 is used as loading control. (E-F) Representative blots showing 17-ODYA labeling of palmitoylated proteins in 2 mice per genotype (out of 3 biological replicates). BM lysates (input) and 17-ODYA–labeled (palmitoylated) proteins were probed with antibodies that detect Ras^G12D (E) or all Ras isoforms (F). Hydroxylamine (HA) sensitivity confirms that 17-ODYA is added to Ras/Ras^G12D by S-palmitoylation. (G) Survival analysis of congenic Nras^G12D (n = 14), Nras^G12D,C181S (n = 12), and WT (n = 5) mice. (H-I) White blood cell (WBC) counts (H) and spleen sizes (I) in 6-month-old animals (n = 5). *P < .05; ***P < .001. Bar graphs show average ± standard error of the mean (SEM).