Figure 5
(A) Results obtained from EMSA and super-EMSA assays where P49 was used as the probe that was interacted with the purified p53 protein. Lane 1 contains the labelled probe. Lane 2 contains the labelled probe and 50 ng of p53 protein. The solid arrow indicates the DNA/p53 complexes. Lane 3 contains the labelled probe, 50 ng of p53 protein and the anti-p53 antibody. The empty arrow indicates the antibody shifted DNA/p53 complexes. Lane 4 contains the labelled probe, 50 ng of p53 protein and 100 times excess of cold P49. (B) Results obtained from EMSA and super-EMSA assays where the P49 probe and nuclear extracts obtained from MCF7/AdrR and MCF7/WT were used. Lane 1 contains the labelled probe. Lane 2 contains the labelled probe and 10 μg of nuclear extract prepared from MCF7/WT cells. The solid arrow indicates the DNA/p53 complexes. Lane 3 contains the labelled probe, 10 μg of MCF7/WT nuclear extract and the anti-p53 antibody. The empty arrow indicates the antibody shifted DNA/p53 complexes. Lane 4 contains the labelled probe, 10 μg of nuclear extract prepared from MCF7/AdrR cells. Lane 5 contains the labelled probe, 10 μg of MCF7/AdrR nuclear extract and the anti-p53 antibody. (C) Chromatin immunoprecipitation assay using MCF7/WT cells treated with 1 μ M DOX. p53–DNA complexes were captured with anti-p53 antibody. Anti-HA antibody was used as the negative control. PCR products representing the WTH3, p21 (positive control) and albumin promoter (negative control) sequences are noted.
