Fig. 5

p73 and Δ133p53 form a complex to upregulate the expression of the DNA DSB repair genes RAD51, RAD52 and LIG4. a Co-immunoprecipitation (IP) analysis of the interaction between p53 or Δ133p53 and p73 under overexpression conditions. 293 T cells were transfected with CMA-p73, CMA-Myc-p53, CMA-Myc-Δ133p53, CMA-p73 plus CMA-Myc-p53 or CMA-p73 plus CMA-Myc-Δ133p53 plasmids. An anti-Myc antibody was used for the IP. The control contained 10% of input from each sample. b Co-IP analysis of the interaction between p53 or Δ133p53 and p73 upon γ-irradiation. HCT116 cells were transfected with siNS or p73i-1, followed with 10 Gy of γ-ray irradiation. The total proteins were sampled at 12 hpi. An anti-p73 antibody was used for IP. IgG was used as a negative control. The control contained 10% of input from each sample. A polyclonal antibody, CM1, was used to detect both p53 and Δ133p53. Middle panel: with a short exposure time; Bottom panel: with a long exposure time. c Relative mRNA expression of the listed genes in Saos2 cells overexpressing p73, Δ133p53 or both p73 and Δ133p53 as measured by quantitative real-time (qRT)-PCR at 12 hpi. Gene expression was normalised against β-ACTIN and expressed as the fold change compared to the vector transfection control. d Western blot analysis of proteins in QSG7701 cells subjected to different treatments as indicated. Protein extracts were analysed via western blotting with appropriate antibodies. e, f The roles of RAD51, RAD52 and LIG4 in the DNA DSB repair pathways, in the context of p73 and Δ133p53 overexpression. Under different conditions, specific siRNAs were used to knockdown RAD51, LIG4 or RAD52 in H1299 cells overexpressing p73 and Δ133p53 together with a HR, NHEJ or SSA reporter construct. Western blot analysis of p73, Δ133p53, RAD51, RAD52 and LIG4 from H1299 cells transfected with different reagents as indicated (e). The average repair frequencies were measured using a qPCR analysis of the repaired assay constructs from three repeat experiments at 24 hpt (f)