Expression levels of oncogenic NRAS and activation of downstream effectors in AML versus CMML mice. (A) Lysates of tumor cells isolated from livers of mice with AML or CMML were run on a 6% to 18% gradient polyacrylamide gel and transferred to a nitrocellulose membrane. The membranes were blotted with anti-Ras and antiactin antibodies to show the expression of oncogenic NRAS with actin as loading control. The ratio of relative expression of 2Xmyc-NRASD12 to that of actin was calculated using the NIH Image 1.63 software. (B) Level of activated Ras in AML and CMML samples was measured by using Raf1-RBD to pull down GTP-bound Ras proteins. (C-D) Activation of effector pathways downstream of Ras was tested in AML and CMML samples by immunoblotting for the indicated proteins. Relative ratios of pS6rp and actin are indicated (D). Samples in lanes 1 to 8 of panels B, C, and D were the same. Lane 9 in panel D is the sample from a vector control mouse.