Fig. 3

DNA-protein interaction in nuclear extracts of ER+ MCF-7 cells in the presence of oligonucleotide carrying palindromic ERE as determined by EMSA. MCF-7 cells were grown in 25 ml of DMEM complete medium (designated as C; DMEM supplemented with 10% FBS, 2.8 µM hydrocortisone, 1 µg/ml insulin, and 12.5 ng/ml of EGF) in 150-mm tissue culture dishes. After 24 hr the medium was replaced with either rich medium (A, lanes 1–4) or with medium containing stripped serum (DCC; DMEM without phenol red, supplemented with 10% dextran-coated charcoal-treated FBS, Hyclone; B, lanes 5–8). The E2 level in this treated serum is lower than 10⁻¹² M. The cells were cultured in DCC medium for an additional 72 hr. Nuclear extracts were made from these cells (40) and DNA-protein complex formation was studied by EMSA under binding conditions described by Kumar and Chambon (24). The reaction mixture in 10 µl contained the indicated amounts of proteins and ³²P-labeled double-stranded oligonucleotide (30,000 cpm, 1 ng) and was incubated at room temperature for 30 min. The reaction was terminated by addition of loading dye. The DNA-protein complex was identified as a retarded radioactive band in the nondenaturing PAGE (6%) system as described previously (41,42). ER-ERE complex and free ³²P-labeled probe are indicated by arrows.