Fluorescently labeled src2

IsoDCA and CDCA were tested for their ability to activate FXR in a cell-free fluorescence resonance electron transfer (FRET) assay using LanthaScreen™ technology according to the manufacturer’s protocol (Thermo Fisher™, PV4833). Briefly, after combining diluted BAs with GST-tagged FXR-LBD, terbium anti-GST antibody and fluorescently-labeled SRC2 in white, flat-bottom 384 well plates (Greiner Bio-one), the reaction was incubated at RT in the dark under gentle shaking (60 rpm) for 1 hour before reading. Fluorescence detection was done in a Tecan Infinite® M100 Pro Microplate reader (Tecan Group, Switzerland) set up according to the LanthaScreen™ Terbium Assay Setup guide available at www.lifetechnologies.com/instrumentsetup. For the first Fluorescence Reading, settings were as follow: Wavelength> Excitation: 332 nm; Bandwith: 20.0 nm/ Emission: 485 nm; Bandwith: 20.0 nm/Flashes> Mode 2 [100 Hz (20)]; Settle time: 0 ms/Mode> Top/Gain> Optimal/Z-position> Calculated from well: (select well with appropriate substrate) /Integration> Lag time: 100 μs; Integration time: 200 μs. After a second Fluorescence Reading was added to the existing protocol, settings were adjusted to the same as described above, except Wavelength> Excitation: 332 nm; Bandwith: 20.0 nm/ Emission: 515 nm; Bandwith: 20.0 nm. Results were expressed as ratio of fluorescence at 520 nm/485 nm.