Immunoblotting reveals that novel mutations confer increased signaling of downstream Ras pathways. (A) Wild-type NRAS and KRAS as well as each mutant were expressed in HEK 293T/17 cells for 48 hours. Whole-cell extracts were subjected to SDS-PAGE analysis and immunoblotted with antibodies specific for total and phospho-MEK and ERK as well as β-actin. (B) Densitometry was performed on blots in panel A. Densitometric units for phosphorylated proteins were normalized to their respective total protein counterparts, and all phospho/total ratios were divided by the ratio for WT N-Ras or K-Ras. Values represent mean plus or minus SEM (n = 3).