FIGURE 9.
The effects of codon 269 mutation on p53 activity in cells; phosphomimetic substitution of serine 269 inactivates p53. The effect of serine 269 mutation on expression of the p53 downstream target genes, Mdm2 and p21, was examined by immunoblotting 20 μg of lysates derived from H1299 cells transfected with pcDNA vector control, wild type p53, p53S269A, or p53S269D mutants using the anti-MDM2 monoclonal antibody 2A10 (A) or the p21 monoclonal antibody Ab-1 (B). C and D, p53 function from reporters containing p53-responsive binding sites. H1299 cells were transfected with the p21 or Bax reporter plasmids alone or with (1–75 ng of) pExpr p53, S269A, or S269D expression vectors. Total DNA was normalized with pExpr vector control plasmid DNA. Twenty-four hours later, cells were lysed, and luciferase activity from p21 (C) or Bax (D) reporters was determined using dual luciferase reporter assays. Data were normalized by expressing luciferase activity (luciferase/Renilla) in relative light units (R.L.U.). The lower panels depict p53 protein levels after transfection by immunoblotting. GAPDH immunoblotting was used as a loading control.