Verification of the isoform-specific shRNA expression vectors. MM.1S cells were transiently transfected with expression vectors for HA-tagged K-, N-, or H-RAS (1 μg/mL, 10 μg/mL, and 2 μg/mL, respectively) in combination with shRNA expression vectors against either K- or N-RAS, or empty pSUPER vector (15 μg/mL). (A) Exemplarily shown are Western blots for wild-type RAS proteins in combination with shRNA expression constructs based on the wild-type RAS alleles. Transfected cell fractions were purified and harvested for Western blotting 2 days after electroporation. The vertical white lines indicate that lanes representing a nonfunctional H-RAS shRNA expression vector have been deleted. (B) Expression of wild-type or mutant (G12A; the mutation present in MM.1S) K-RAS in MM.1S cells. Although allele-specific shRNA expression vectors may show slightly better efficiency with their perfect match, both lead to effective knockdown of mutant or wild-type K-RAS, rendering mutation specific knockdown impossible. β-Actin served as loading control.