Fig. 2.

Analysis of the DNA binding specificity of p53. (A) Three sequences previously reported to bind p53 were analyzed by PLA along with specificity controls in nuclear lysates prepared from MCF-7 cells. Positive controls: Pos I was a p53 consensus sequence published by Kastan et al. (23), Pos II (24) was a focal adhesion kinase promoter region shown to interact with p53, and Pos III was a polymorphic microsatellite that mediates induction of p53-inducible gene 3 by p53 (25). Negative controls: Pos I mut was a probe in which four nucleotides within the consensus binding site had been altered, and Pos I inhib control, a 1,000-fold excess of the consensus p53 DNA probe without the proximity probe extension, was added to the reaction together with the full-length consensus probe. The negative control was a probe positive for HNF-4α binding but with no known affinity for p53, and in the no lysate control, no nuclear lysate was added to the reaction. The S/N is shown on the y axis. (B) Dilutions of MCF-7 nuclear lysate were analyzed with the Pos I (filled bar) and the Pos I Mut probe (open bar) to determine the limit of detection of p53 in the extract.