Dual Luciferase Assay for FKBP12 Variants

Dual luciferase assays were performed using 293FT FKBP12^WT-Nluc and FKBP12^F36V-Nluc cells^{6 (link)}. In brief, cells were plated at 2000 cells per well in 20 µL of appropriate media in 384-well white culture plates (Corning), allowed to adhere overnight, and 100 nL of compounds were added using a Janus Workstation pin tool (PerkinElmer) for 24 h at 37 °C. To evaluate Fluc signal, plates were brought to room temperature, 20 µL of Dual-Glo Reagent (Promega) was added for 10 min and luminescence was measured on an Envision 2104 plate reader (PerkinElmer). Subsequently, 20 µL of Dual-Glo Stop & Glo Reagent (Promega) was added for 10 min and luminescence was again measured to capture Nluc signal. DMSO-normalized ratios of Nluc/Fluc signal was analyzed and plotted using GraphPad PRISM v8^{6 (link)}.