Figure 8. Validation for thermal shift experiments.

(A) FXR agonist GW4064 robustly stabilizes the FXR-LBD at concentrations ≥100 μM (ratio to protein ≥20:1) and produces a significant thermal shift. (B) GW4064 showed no interaction with the dye SYPRO Orange. (C) GW4064 produced a robust thermal shift of about 6 °C and with our conditions (5 μM FXR-LBD) had an EC₅₀ value of 60 ± 4 μM. (All experiments were repeated independently four times in duplicates and yielded uniform results; Fig. 7A,B represent one repeat).