Figure 2.KRAS insertion mutants activated RAS signaling by enhancing cellular accumulation of active RAS (RAS-GTP) and activating p-ERK. NIH3T3 and 293FT cells were transfected with KRAS mutants, and RAS-GTP protein in the cell extract were immunoprecipitated with agarose beads containing Ras binding domain of Raf-1. Protein levels in both whole cell extracts (pan-RAS and pERK) and precipitated samples (RAS-GTP) were analyzed by western blot analysis as indicated. Representative results from 3 independent experiments were shown.