The ability of TB to either activate GR-mediated transcription or to inhibit dexamethasone -induced GR activity, was examined as previously described using CV-1 cells cotransfected with 1 μg of pCMVhGR and 5 μg MMTV-luciferase reporter ( Parks et al ., 2001 ). Briefly, cells were plated at 200,000 cells/60 mm dish and transfected using 5 μl Fugene reagent (Boehringer Mannheim) plus 95 μl serum-free medium per dish using the manufacturer’s protocol. Twenty-four h after transfection, duplicate wells were treated with either vehicle only, 1 nM Dex only, or increasing concentrations of TB both with and without Dex cotreatment in Dulbecco’s Modified Eagle Medium (DMEM) with 5% Dextran coated charcoal stripped fetal bovine serum (DCC FBS). After 24 h of exposure to test chemicals, cells were washed and harvested with ml cell lysis buffer. Luciferase activity was detected using ml of cell lysate, and relative light units were determined using a Monolight 2010 luminometer (Analytical Laboratories).
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