Assay Method Information

Assay Name:  Transactivation Assay
Description:  PC-3 cells (Kaighn et al., Invest. Urol. 17: 16-23, 1979) were plated out at a density of 10000 cells per well of a 96-well cell culture plate in RMPI 1640 medium (F1235, Biochrom AG Berlin, Germany) that contained activated charcoal-treated calf serum (FCS Serum Gold, PAA Laboratories) at a concentration of 5% (v/v). On the next day the cells were transiently transfected with the pSG5-vector (#216201 Stratagene), which contained the sequence of the androgen receptor mutant E709Y (Georget et al., Mol. Endocrinol. 20(4): 724-734, 2006), and with the MMTV-luciferase plasmid (see above, transactivation assay for the androgen receptor mutant W741C). The cells were treated with the test substances in concentrations from 1×10^−8 to 1×10^−10 M in the presence of 1×10^−10 M R1881 and were incubated overnight at 37° C. and 5% CO2. After 24 hours, 100 μl of Steady Glo Lysis and Detection reagent (E2550, Promega) was added per well and the luminescence was read in a Victor3 Luminometer (PerkinElmer) for 1 second per well. The luminescence values obtained were normalized, wherein 100% corresponded to the effect of the unstimulated control (without R1881), and 0% corresponded to the effect of the stimulated control (R1881 plus DMSO instead of test substance).
Affinity data for this assay

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