Generation and validation of BRAF knockout cell lines. A: Schematic representation of the knockout procedure resulting in recombination of BRAF exon 15 and substitution by a resistance cassette. B: Genealogy of the corresponding tumor cell clones. From the parental colorectal cancer cell line RKO a single clone was generated by limiting dilution. Subsequently, a first oncogenically mutant allele (onc) was deleted by infection with AAV-BRAF-Hyg virus and the cell line RBOW (RKO-derived clone BRAF-/onc/wt) was established. In a secondary targeting using AAV-BRAF-Neo virus either the second V600E allele or the wild-type allele (wt) was targeted to generate RBO (RKO-derived clone BRAF-/-/onc) and RBW (RKO-derived clone BRAF-/-/wt) clones. Genotypes were validated by A/T peak ratio in sequencing histograms. Microscopy scales: 250 μm × 150 μm. C: Aliquots of RKO-E1 and knockout cell clones were incubated under different serum conditions, subsequently lyzed, and used for Western blot analysis.