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The wild-type BRCA1 expression plasmid (wtBRCA1) was created by cloning the BRCA1 cDNA into the pcDNA3 vector (Invitrogen) with artificially engineered 5′ Hin dIII and 3′ Not I sites. For transcription assays, we incubated asynchronously proliferating cells overnight at 50% to 70% of confluency in 24-well dishes with 0.5 μg of each vector (except where otherwise indicated) in serum-free Dulbecco's modified Eagle's medium (DMEM) containing Lipofectamine (Life Technologies). Cells were washed, incubated in serum-free phenolphthalein-free DMEM (0.2 ml per well) with or without 17β-estradiol (1 μM) for another 24 hours, and harvested for luciferase assays. Values are means ± SEM of four replicate wells and are representative of at least three experiments. Results were similar whether ER-α assays were performed in serum-free DMEM or in the presence of 2% charcoal-stripped serum.
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Supported by U.S. Public Health Service (USPHS) grant R01-ES09169 (E.M.R.), the Elsa U. Pardee Cancer Foundation of Michigan (E.M.R.), the Susan G. Komen Breast Cancer Foundation (R.G.P.), and USPHS grant RO1-CA75503 (R.G.P.).
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