RAF-1 pull-down assay reveals that novel mutations confer increased Ras-GTP. (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 incubated with RAF-1 peptides conjugated with agarose beads for precipitation of Ras-GTP. Precipitates were subjected to SDS-PAGE analysis and immunoblotted with an antibody specific for total Ras. (B) Densitometry was performed on blots in panel A. Densitometric units for Ras-GTP were normalized to their respective total Ras counterparts, and all Ras-GTP/total Ras ratios were divided by the ratio for WT N-Ras or K-Ras. Values represent mean plus or minus SEM (n = 3).