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. Author manuscript; available in PMC: 2015 May 18.
Published in final edited form as: J Biomol Screen. 2011 Feb;16(2):183–191. doi: 10.1177/1087057110389040

Figure 2. RORα-PGC-1α cross-titrations and validation of His-RORα-LBD protein conformational change.

Figure 2

A) Increasing concentrations of Biotin-PGC-1α NR1 peptide (8.4 pM-500 nM) were incubated with a constant amount of 25, 50, 75, and 100 nM, respectively of His-RORα-LBD as described in Material and Methods and table shows Hill Slope, EC50 (M), S/B, and Z’ for the indicated His-RORα-LBD concentrations. B) Increasing concentrations of His-RORα-LBD (8.4 pM-166 nM) at a constant amount of 70 nM of Biotin-PGC-1α NR1 (EC90 value) in a 15 µL assay volume. The final concentration of each bead was 12.5 µg/mL. Plates were incubated for 1h at room temperature. Each data point is represented as the mean ± S.D. of 3 independent experiments (N=3). C) Increasing concentrations of compound 7α-hydroxycholesterol (425 pM-25 µM) were pre-incubated with His-RORα-LBD (75 nM) and both acceptor and donor beads for 1h at room temperature. The peptide Biotin-PGC-1α (30 nM) was added to each well and further incubated for 2h at room temperature. The final concentration of each bead was 30 µg/mL in 20 µL final volume. Data are represented as the mean ± S.D. of 2 independent experiments (N=7). The EC50 values were calculated by non-linear regression analysis (sigmoidal dose-response-variable slope) using GraphPad Prism.