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6 transfected cells in Dulbeco's Modified Eagle Media (Mediatech, Inc.) in 100 μL in 96 well plates. Bound ligand was separated from unbound ligand by filtration using a Filtermate (Packard) and total counts bound determined on a TopCount NTX reader (Packard). Standard error of the assay day-to-day was <30%; well-to-well variability was <10%.
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18644372803
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Castanedo G.M., Sailes F.C., Dubree N.J.P., Nicholas J.B., Caris L., Clark K., Keating S.M., Beresini M.H., Chiu H., Fong S., Marsters Jr. J.C., Jackson D.Y., and Sutherlin D.P. Bioorg. Med. Chem. Lett. 12 (2002) 2913
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67649907158
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1H NMR.
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26
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67649931217
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3OH).
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67649924185
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2O requires 426.5.
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67649907157
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Protocol for cytotoxicity assay: HeLa cells were seeded at 5 k/well in a 96 well plate (6 plates-for triplicate 0 and 72 h readings). A threefold dilution series of each compound was generated, starting at 10 mM. The solution was diluted eight times to prepare 9 concentrations ranging from 10 mM to 150 nM. Compound was added to cells (1-100 μL total volume), and the final concentration of compound was 100 μM to 15 nM. Zero and 72 h time points were recorded as follows: (a) 10 μL Alamar Blue reagent was added to the wells, and the samples were incubated at 37 °C for 3 h; (b) fluorescence intensity was then recorded on an LJL Analyst. The relative growth was compared to a DMSO control well for each compound.
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67649942804
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3H]Inositol (82.0 Ci/mmol, Amersham Pharmacia Biotech) overnight at 37 °C. Test compounds were then added in inositol-free DMEM containing 0.3% BSA (Sigma) and 10 mM LiCl (Sigma) for 60 min at 37 °C. The cells were lysed with 20 mM formic acid for 2 h at 4 °C and added to RNA-binding Ysi scintillation proximity assay beads (Amersham Pharmacia Biotech) for 30 min. Plates were stored overnight at room temperature in the dark and read the next day on a TopCount NTX.
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