The FKBP:FRB model system was used with HEK293 cells transfected with either NanoBiT or firefly luciferase fusion constructs. After overnight incubation cells were treated with rapamycin to stimulate FKBP:FRB interaction. Panel A. Cells were treated with rapamycin as indicated and incubated at room temperature (21°C) two hours followed by reagent addition and luminescence measurement. Panel B. After reagent addition, cells were incubated at 37°C and luminescence measured for approximately 30 minutes. Cells were then treated with 30nM rapamycin and luminescence monitored for an additional 40 minutes.