a, Western blot analysis of p53 protein levels in untreated or doxorubicin-treated (0.2 μg ml−1 Dox) p53−/−, p53+/−, p5325,26,53,54/− and p5325,26,53,54/+ MEFs. β-Actin served as a loading control. b, Western blot analysis of anti-Flag immunoprecipitation from p53−/− MEFs transiently overexpressing HA–p53 and Flag–p53 or Flag–p5325,26,53,54. HA–MBP and Flag–eGFP were used as negative controls. Immunoprecipitated protein and 10% input were probed with either anti-HA or anti-Flag antibodies. (The microgram ratio of HA–p53 to Flag–p53 or Flag–p5325,26,53,54 plasmid DNA was 1:1 or 1:2.5, as indicated above the blot (see Fig. 3b). c, Heat map examining the transactivation capacity of p5325,26,53,54 on p53-dependent genes identified by microarray analysis through comparison of six HrasV12; WT p53 MEF lines with six HrasV12; p53-null MEF lines, as previously described3. Three independent HrasV12; p5325,26,53,54/25,26,53,54 MEF lines were analysed, and the gene expression profiles were indistinguishable from those of HrasV12; p53-null cells. The numbered columns indicate independent MEF lines. Blue denotes repressed genes, and red denotes induced genes. d, qRT–PCR analysis of p53 target gene expression in untreated MEFs derived from p53+/+ and p5325,26,53,54/+ E13.5 embryos. Graphs indicate the mean ± s.d. from four independent MEF lines after normalization to β-actin gene expression. **, P < 0.01; ***, P < 0.005; Student’s t-test. e, qRT–PCR analysis of p53 target gene expression in p53+/+ and p53−/− MEFs stably transduced with empty vector, Flag–p53 or Flag–p5325,26,53,54. The representative gene expression from one experiment is shown: mean ± s.d. of technical triplicates after normalization to β-actin gene expression. The experiment was performed in duplicate.