Figure 5.

Disruption of a critical NRD-DBD interface hyperactivates B-Myb. HCT116 cells were transfected with a luciferase reporter plasmid containing three Myb-binding sites (MBSs) upstream of a minimal promoter together with plasmids expressing Flag-tagged wild-type (WT) B-Myb or the indicated mutants. DBDdel is mutant with entire DNA-binding domain deleted (amino acids 12-182). Mean values ± SD of four biological replicates are given, and significances were calculated by the Students paired t test (∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001 compared with WT B-Myb). Expression levels of B-MYB variants in the luciferase assay samples were analyzed by SDS-PAGE/Western blot. DBD, DNA-binding domain; NRD, negative regulatory domain.