Fig. 4

ER and progesterone receptor (PR) status in breast cancer biopsy specimens. Estradiol specific receptor (ER) and (PR) levels in extracts of frozen human breast tumor biopsy specimens were measured by (A) ³H-E2 binding using the hydroxylapatite method (32). 10 µg (protein) of nuclear extract from cell lines or 20 µg of tissue extracts were used for this assay. (B) shows the integrated intensity of the immunoreactive signals generated by the specific interaction of anti-ER or anti-PR antibody with ER or PR. Each reaction of duplicate samples with the immunodetection assay contained 20 µg of tissue extracts, 1 ng nonradioactive double-stranded estradiol response element (ERE) oligonucleotide, plus other components, and was incubated under conditions described in “Materials and Methods.” Control reactions contained the same amount of bovine serum albumin (BSA) instead of tissue extracts. Nuclear extracts (10 µg) from ER-positive [ER (+)] T47D cells and ER-negative [ER (−)] MDA-MB-231 human breast cancer cell lines were analyzed similarly and served as positive and negative controls, respectively. (C) shows the integrated intensity of the autoradiographic signals generated by ³²P-ERE-ER or ³²P-PRE-PR complexes immobilized on the nitrocellulose membrane as described in “Materials and Methods.” The reactions were run in duplicate under conditions described in the legend to (B), with the exception of the use of double-stranded ³²P-ERE-oligonucleotide or ³²P-PRE (1 ng, 30,000 cpm) replacing the nonradioactive oligonucleotides.