Figure 4

Id4 promotes DNA binding and transcriptional activity of wild type and mutant p53. A. EMSA with p53 consensus DNA binding response element with nuclear extracts from LNCaP (L), LNCaP-Id4 (L-Id4), DU145 (D), DU145 + Id4 (D + Id4) and PC3 cells. Nuclear extracts from PC3 cells, null for p53 and LNCaP cells with wild type p53 were used as negative and positive controls respectively for binding to wild type p53 response element. Excess unlabeled (EU) wild type p53 response element was used to monitor non-specific binding. The biotin labeled mutant p53 response element (mt) incubated with nuclear extracts from LNCaP cells (L + mt) was used to demonstrate specificity of EMSA. B. Quantitative p53 DNA binding in a sandwich ELISA based system. P53 was captured by double stranded oligonucleotide with p53 response element immobilized on a 96 well plate. The captured p53 was detected using p53 antibody by measuring the intensity at 450 nm using HRP coupled secondary antibody. C. The p53 transcriptional activity as determined by transiently transfecting cell lines as indicated above with p53 response element driven luciferase reported plasmid (wt-p53RE). The data is normalized to Renilla luciferase. The mutant p53 luciferase reporter plasmid was used as a negative control (mt-p53RE). The p53-luciferase reporter activity in LNCaP-Id4 (L-Id4) was normalized to LNCaP (L) and that of DU145 + Id4 (D + Id4) with DU145 (D). The data from 3 different experiments in triplicate is expressed as mean + SEM (*: P < 0.001).