TMZ induces AKT phosphorylation, IκB-α degradation, and RelA nuclear translocation in MMR-proficient but not in MMR-deficient cells. HCT116/3-6, HCT116 and M10 cells were cultured in the presence of 50 μM TMZ+10 μM BG, or BG alone as a control, for 48 and 72 h (A, B) or 72 h (C). Total cell extracts (A, B) or nuclear extracts (C) were resolved on 10% SDS polyacrylamide gels, transferred to nitrocellulose membranes and probed with antibodies against the indicated proteins. Anti-actin mAb or anti-lamin A/C mAb were used for equal loading control. The immune complexes were visualized using ECL. For the indicated samples, the densitometric levels of phospho-AKT, IκB-α and RelA were normalized to the respective levels of actin or lamin. Fold changes of protein expression in TMZ-treated samples were then calculated with respect to the protein levels in the corresponding controls, to which the arbitrary value of 1.0 was assigned. The results are representative of three (A, B) or two (C) independent experiments.