Fig. 1

Nitrocellulose filter binding assay (NMBA) for identification of ERE-ER and PRE-PR complexes. MDA-MB-231, 21PT, MCF-7, and T47D cells were grown in rich medium under standard tissue culture conditions and nuclear extracts were prepared as described by Dignam et al. (25). (A) The total specific receptor (ER) and progesterone receptor (PR) level were measured with NMBA by using the immunodetection assay with nonradioactive estradiol response element (ERE) or progesterone receptor response element (PRE) and (B) the DNA bound fraction by using ³²P-labeled ERE/PRE in the reaction mixture. Immunodetection was carried out by using anti-PR/ER antibody as primary and anti-mouse imunoglobulin G-horse radish peroxide (IgG-HRP) conjugate as secondary, followed by enhanced chemiluminescent protein (ECL) detection analysis as described above. Binding reactions were carried out in a 96-well plate containing 10 µg of nuclear protein, double-stranded ³²P-ERE (1.0 ng and 30,000 cpm) or the same amount of ³²P-PRE (B) and binding buffer. Following incubation at room temperature for 30 min, the samples were applied to a nitrocellulose membrane in a slot blot assembly system. 15 min later, the membrane was washed three times with binding buffer. Control reactions with no extract (indicated as None, B, row 1) or 10 µg of nonspecific protein, such as bovine serum albumin (BSA), were run to determine nonspecific binding of free ³²P-labeled oligonucleotides to nitrocellulose membrane (B, row 2). A typical result of three such experiments is presented.