2.5. Hsp90/ Cdc37 Split Renilla Luciferase Assays

HEK cells were seeded in a 10 cm dish and transfected the next day with 10 µg plasmid pcDNA3.1(+)-NRL-Hsp90, pcDNA3.1(+)-NRL-N-Hsp90, or pcDNA3.1(+)-Cdc37-CRL using jetPRIME. After 48 h, cells were harvested and lysed in 1 mL 1 × lysis buffer provided in the Renilla Luciferase Reporter Assay System Kit from Promega (cat. E2820) and incubated on ice for 10 min. Cell lysates were then cleared by centrifugation at 13,000× g for 1 min. Then, 20 µL assay buffer from the aforementioned kit was added to each well of a white 96-well flat-bottom plate (cat. 3917, Costar, Corning Inc., Corning, NY, USA). After that, test compounds or 0.1% DMSO control, as indicated, were added to each well. Then, 5 µL NRL-Hsp90- or NRL-N-Hsp90-lysate was added to each well one row at a time. Then, the plate was incubated for 5 min at RT. After that, 5 µL Cdc37-CRL was added and incubated for 2 min at RT. Just before reading, 20 µL assay buffer containing 2 × Renilla luciferase substrate (coelenterazine) was added and the plate was read using a Synergy H1 Hybrid Multi-Mode reader (BioTek) in the luminescence detection mode, as described previously in detail [23].