CHIP assay to detect the ability of MDM2 to bind to the p65 promoter in vivo.

(A) Flow chart of the experimental design. (B) Agarose gel electrophoresis shows the PCR results from each CHIP assay. MDM2 expression plasmid and either wt p65F1 (lane 2) or mutant p65F1xx (lane 5) were cotransfected into EU-4 cells and precipitated with anti-MDM2 antibody. The p53 expression plasmid and wt p65F1 were also cotransfected into EU-4 cells and precipitated with anti-p53 antibody (lane 8) to detect the ability of p53 to bind to the p65 promoter. For negative control, immunoprecipitation was performed using a normal mouse IgG, or it was performed in the absence of antibody (no) in each experiment (lanes 3, 6, 9 and 4, 7, 10, respectively). Lane 1 shows DNA size markers. PCR product (379 bp) in lane 2 contains the first 2 Sp1-binding sites in the p65 promoter.