Western analysis of oncogenic RAS signaling. Knockdown of K-RAS in MM.1S cells (harboring an activating G12A K-RAS mutation) or N-RAS in JJN-3 cells (harboring a Q61K N-RAS mutation) led to strongly decreased levels of phosphorylated ERK1/2, indicative of involvement of the RAS/MAPK pathway in oncogenic RAS signaling. Phosphorylation levels of Akt and of its substrates FOXO1/3a and GSK-3β were not significantly affected (shown for MM.1S cells only). Cells were transfected with shRNA expression vectors (20 μg/mL) designed against either the mutant K-RAS allele (but which also leads to knockdown of the wild-type protein), or against both N-RAS alleles (the targeted sequence does not match the position of the mutation at codon 61), purified and harvested for Western analysis 2 days after transfection. β-actin served as loading control.