Figure 2. Protein DNA interactions at the sequences overlapping rs378854.

(A) Electrophoretic mobility shift assays (EMSA) were carried out with oligonucleotides containing the common (c) and minor (m) alleles of rs378854 and nuclear extracts from the breast cancer cell line MCF-7 and the prostate cancer cell line PC3. Competitor oligonucleotides were present at 100-, 30- and 10-fold excess for MCF-7 and 30-fold excess for PC3 and are indicated above the lanes. The red and black closed arrows denote the YY1 and SP1 complexes, respectively. (B) EMSAs of the oligonucleotide containing the minor allele were carried out with PC3 nuclear extract and 30-fold excess of competitor oligonucleotides as shown. (C) Supershift of the complex using polyclonal antibody against YY1, SP1, Oct-1 and C/EBPα with PC3 nuclear extracts. The red and black open arrows denote the YY1 and SP1 supershift complexes. (D) An alignment of the two alleles with a YY1 binding site [28] is shown. (E) Chromatin immunoprecipitation assay showing the fold-enrichment of the S-DHS and the positive control (glucocorticoid receptor, GR) sequences relative to a negative control (S-DHS-ve) after immunoprecipitation with YY1 antibody in MCF-7 breast 1542-CP prostate cancer cells.