Gaussia flex luciferase kit

The LUMIER assay was performed essentially as described previously (Taipale 2014). Briefly, 3X FLAG-tagged bait proteins (table S2) were transiently transfected in 96-well format into a 293T cell line stably expressing HSF1 with a codon-optimized Renilla reniformis luciferase C-terminal tag. Two days after transfection, cells were rapidly washed in 1× ice-cold phosphate-buffered saline (PBS) and lysed in ice-cold HENG buffer (50 mM HEPES-KOH pH 7.9, 150 mM NaCl, 2 mM EDTA, 5% glycerol, 0.5% Triton X-100 supplemented with protease, and phosphatase inhibitors) (Taipale 2012). The lysate was transferred into 384-well plates coated with monoclonal M2 antibody and blocked with 3% bovine serum albumin/5% sucrose/0.5% Tween 20. Plates were incubated at 4°C for 3 hours, after which plates were rapidly washed in HENG buffer. Last, luciferase-tagged HSF1 was detected by measuring luminescence using the Gaussia FLEX Luciferase Kit [New England Biolabs (NEB)]. An HSF1-LUMIER interaction score was determined from the log₂-transformed bait-FLAG/GFP-FLAG luminescence ratio (reported in table S2).