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Fig. 7. p53 binds to the PIK3CA promoter and suppresses its activity. (A) ChIP results for OSEC2 cells. Antibodies used for immunoprecipitation (IP) are indicated below the panels. OSEC2 cells at 34°C have reduced p53 activity, whereas the cells at 39°C have full activity. PCR amplification with GAPDH primers of samples immunoprecipitated with RNA polymerase II antibody is presented as a positive control. Input DNA acts as control for levels of DNA present in each sample. Amplification with primers around the known p53-binding sites on the p21 promoter were used as positive controls. (Ba) An electrophoretically retarded complex (shift) was formed with recombinant p53 and biotin-labeled oligo4. Formation of this complex was inhibited with an excess of unlabeled wild-type oligo4. However, the unlabeled mutant oligo4, with point mutations in the core (CnnG) binding region, did not compete with the biotin-labeled oligo4 to the same extent. In addition, the biotin-labeled mutant oligo4 showed reduced interaction with p53 compared to wild-type oligo4. (Bb) Similarly to the recombinant p53, the use of nuclear lysates from OSEC2 cells at 39°C with p53 resulted in the formation of an electrophoretically retarded complex, which was competed with excess unlabeled wild-type oligo4. Similarly, the unlabeled mutant oligo4 did not compete with the biotin-labeled oligo4 to the same extent; and the biotin-labeled mutant oligo4 showed reduced interaction with p53 compared with the wild-type oligo4. The use of nuclear lysates from OSEC2 cells at 34°C without p53 did not lead to formation of an electrophoretically retarded complex (shift). Addition of an anti-p53 antibody (PAb421) to the reaction mixture induced a supershift of the protein-DNA complex, indicating the specificity of oligonucleotide for p53. (C) OSEC2 cells transfected transiently with promoter1a construct (pGL3-P1A) showed 83% less luminescence (PIK3CA-promoter activity) at 39°C compared with at 34°C. pGL3-P1A-mut4 construct showed significantly less decrease in promoter activity after the switch in temperature to 39°C (less than 50% decrease). pGL3-control was used as positive control and pGL3-basic was used as negative control. The values presented were normalized with the internal control (phRL-TK).
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