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64249093433
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note
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The ER competitor assays were performed according to the manufacturer's (Invitrogen, Carlsbad, CA) recommendations with some modifications. Human recombinant ER was used at the recommended concentration of 15 nM and the Fluormone™ ES2 was used at a concentration of 1 nM. Aliquots (20 μL) of the mixture of ER and Fluormone™ ES2 were distributed in 384-well, black flat-bottom plates and serial dilutions of test compounds in DMSO were added. Each compound was tested in duplicate, and 1 μM E2 was used as a positive control. The DMSO concentration was kept at 1% (v/v) throughout the experiment. After 2 h, the fluorescence polarization (FP) was measured using an Analyst™ AD & HT Assay Detection Systems reader (Molecular Devices, Sunnyvale, CA) equipped with 485 nm excitation and 530 nm emission filters with the appropriate FL505 dichroic mirror. Data was then analyzed by linear regression using GraphPad Prism's one-site competition method. The fit was constrained by the high polarization control, which was the ER/ES2 complex with no competition, and the low polarization control, which was the ER/ES2 complex with 1 μM E2 for 100% competition.
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20
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13944255737
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21
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64249119928
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note
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50 values.
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22
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24
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64249159305
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note
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3 cells/well and allowed to adhere for 72 h in phenol red-free RPMI-1640 containing 10% FBS. Test agents were added (concentrations ranging from 3.2 nM to 50 μM) and cells were incubated for 72 h. Cell density was determined using the MTS assay as described above.
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27
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0034727853
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