FIG. 2.

p53 upregulated by Mdm2 siRNA binds to the response elements in the p21 promoter both in vitro and in cells. U2OS cells were transfected with Mdm2 siRNA oligonucleotides or treated with 0.1 μg/ml doxorubicin (DOX) for 24 h. (A) Volumes of the different extracts containing similar amounts of p53 were used in oligonucleotide pull-down assays to measure binding to the p21 5′ biotinylated probe or a control probe (cont.). 1x and 2x represent two levels of whole cell extracts (wce), and “input” corresponds to 10% of those amounts. The presence of p53 in the precipitated complexes was detected by immunoblotting. (B) Cells were cross-linked, and extracts were subjected to ChIP analysis. Radioactive PCR was performed to detect the occupancy by p53 of the response elements in the p21 promoter. A nonrelated genomic region was amplified as a control. (C) p53 levels in the extracts used for ChIP were assayed by immunoblotting.