(A) Il10^–/– and Il10^–/–Egfr^wa5 colon tumors have a single G→T point mutation in Braf encoding a Q609H substitution in the highly conserved activation domain (human numbering noted). The V600E mutation (*) is commonly observed in human colon cancer. (B–E) YAMC cells were stably transduced with the indicated Braf mutations. (B) BRAF expression in each line, as determined by in-cell Western; cell labeling with IRDye 800 NHS ester was used as a loading control; n = 3. (C) Basal levels of ERK phosphorylation, as determined by in-cell Western; total ERK was used as a loading control; n = 6; NS, P > 0.05, *P < 0.05 versus Braf^WT. (D) Braf^Q609H increases unstimulated cellular proliferation. Proliferative rate was determined by EdU incorporation in cells treated for 24 hours with control medium, 10 μM U0126, or 10% fetal bovine serum. EdU incorporation was determined following a 2-hour pulse and quantified as the percentage of EdU-positive nuclei, and NuclearMask was used to stain nuclei; n = 6; NS, P > 0.05, ***P < 0.001 versus control-treated cells. Representative images of control Braf^WT and Braf^Q609H cells are shown. Original magnification, ×10. (E) Braf^Q609H inhibits basal and TNF + IFN–induced apoptosis. Apoptotic rate was determined by caspase-3 activity assay in cells treated for 5 hours with control medium, 10 μM U0126, or 100 ng/ml TNF + 200 U/ml IFN. Caspase-3 activity was determined in live cells and quantified by the degradation of a specific caspase-3 substrate (CSP3); n = 3–6; NS, P > 0.05, **P < 0.01, ***P < 0.001 versus control-treated cells. Representative images of control Braf^WT and Braf^Q609H caspase-3 degradation product are shown; DRAQ5 was used a DNA dye loading control.