(A and B) Recombinant wild-type or p53C275,277A mutant protein (10 μM) was incubated with hemin (50 μM) on ice, followed by gel filtration and TMB assay (Pierce).
(C) A close-up view of the C275 and C277 residues of human p53 in the p53 core domain-DNA (Cho et al., 1994) (derived from PDB 1TSR).
(D) EMSA of the indicated hemin amounts, p53 protein, and double-stranded DNA probes containing p53RE consensus sequences. Band designations are free DNA (F) and bound DNA (B; p53-DNA complex).
(E) HCT116 p53−/− cells transiently expressing HA-tagged wild-type p53 were cotransfected with indicated luciferase reporter plasmids, then treated with or without hemin (10 μM, 6 hr). Error bars indicate ± SEM (n = 3). **p < 0.01.