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. 2019 Jul 11;294(35):12957–12974. doi: 10.1074/jbc.RA119.008100

Figure 3.

Figure 3.

p53 binds to the p53REs of the 5′-upstream regulatory region and coding region of KAISO. A, structure and oligonucleotide pulldown/Western blotting assay of p53REs of the KAISO gene. Endogenous p53REs of KAISO 5′-upstream regulatory region (ChIP#1, bp −4354 to −4254), intron 1 (ChIP#2, bp +2426 to +2556), and exon-coding sequence region (ChIP#3, bp +2885 to +2982) are shown above. Arrows, locations of the ChIP oligonucleotide primer sets spanning the p53-responsive elements (p53REs). Probe #1, #2, #3, and #4 represent the p53REs used for oligonucleotide pulldown assays. Shown is an oligonucleotide pulldown/Western blotting assay of p53 binding to the p53REs of KAISO 5′-upstream regulatory region and exon 2 coding sequence region. H1299 p53-null cell extracts with ectopic p53 were incubated with biotinylated double-stranded oligonucleotides. The mixtures were further incubated with streptavidin-agarose beads and precipitated by centrifugation. The precipitate was analyzed by a Western blotting assay (WB) using antibodies against p53 or GAPDH. B, ChIP assays of p53 binding to a potential p53RE of the endogenous KAISO 5′-upstream regulatory region and coding region. H1299 p53-null cells were transfected with a p53 expression vector and immunoprecipitated (IP) with anti-p53 antibody, followed by PCR amplification of the region flanking p53RE1, p53RE2, and p53RE3. **, p < 0.01. Error bars, S.D.