EMSA to examine the binding of MDM2 to the first Sp1 site of the p65 promoter.
Nuclear extracts from UOC-B11 cells and MDM2 and control plasmid-transfected EU-4 cells (EU-4 MDM2 and EU-4 neo) were incubated in binding reactions with annealed and 32P-labeled wt or mutant (mut) oligonucleotide probe containing the first Sp1-binding site of the p65 promoter. Samples were run on a nondenaturing 5% polyacrylamide gel and were imaged by autoradiography. Lane 1, labeled wt probe without nuclear extracts; lanes 2 to 5, labeled wt probe incubated with nuclear extracts of EU-4 MDM2; lane 6, labeled mut probe with nuclear extracts of EU-4 MDM2; lanes 7 to 10, labeled wt probe with nuclear extracts of EU-4 neo cells; lanes 11 to 14, labeled wt probe with nuclear extracts of UOC-B11 cells. In reactions depicted in lanes 3, 8, and 12, 25-fold molar excess of nonlabeled wt oligonucleotide was added. In reactions depicted in lanes 4, 9, and 13, cell extracts were preincubated with 2 μg anti-MDM2 antibody for 1 hour at 4°C before probes were added. In reactions depicted in lanes 5, 10, and 14, cell extracts were preincubated with 2 μg anti-Sp1 antibody. Specific protein–DNA complexes and supershift with anti-MDM2 and anti-Sp1 antibodies are indicated.
