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Day, C. L.; Chen, L.; Richardson, S. J.; Harrison, P. J.; Huang, D. C. S.; Hings, M. G. J. Biol. Chem. 2005, 280. High-throughput FP assay: Chemical inhibitors were dissolved in 10% DMSO at 100 μM stock concentration. From this solution, 4 μL were incubated with 8 μL of Bfl-1 working solution (7.4 nM GST-Bfl-1 fusion protein in 25 mM Bis-Tris pH 7.0, 1 mM TCEP, 0.005% Tween 20) at room temperature away from direct sunlight in 384-well black plates (Greiner Bio-One, Monroe, NC). After 1 h, FITC-Bid BH3 peptide (8 μL of 5.6 nM in 25 mM Bis-Tris pH 7.0, 1 mM TCEP, 0.005% Tween 20) was added, and plates were incubated at room temperature away from direct sunlight. Fluorescence polarization was measured using an Analyst GT plate reader (Molecular Devices, Inc., Sunnyvale, CA) using fluorescein filters (excitation filter 485 nm, emission filter 530 nm, dichroic mirror # 505 nm). Data analysis was done using CBIS software (ChemInnovations, Inc., San Diego, CA). Dose-response confirmation: Chemical inhibitors were serially diluted in DMSO then in water so final concentration was in 10% DMSO. Ten concentrations of each compound were used. Each inhibitor (4 μL) was transferred to 384-well black plates (Greiner Bio-One, Monroe, NC) followed by GST-Bfl-1 solution described above (8 μL). Plates were incubated for 1 h at 4 °C. FITC-Bid BH3 peptide solution described above (8 μL) was added, and plates were incubated for 4 h at room temperature. Fluorescence polarization was measured at described above. Data analysis was performed using sigmoidal dose-response equation through non-linear regression.
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77958037981
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note
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50 data in the tables) can be found at: http://graphpad.com/help/prism5/prism5help.html? usingstatistical-analyses-step-by-s.htm.
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http://www.ncbi.nlm.nih.gov/pccompound.
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