NanoBiT luciferase studies

The NanoBiT assay utilizes a structural complementation-based approach to monitor protein–protein interactions within living cells. Large BiT (LgBiT; 18 kDa) and Small BiT (SmBiT; 1 kDa) subunits of NanoLuc Luciferase were optimized for the analysis of protein interaction dynamics. When LgBiT and SmBiT subunits are separated, the Large BiT part loses the majority of luciferase activity. However, when the direct interaction between fusion proteins on LgBiT and SmBiT occurred, the interaction promotes structural complementation between LgBiT and SmBiT and results in full luciferase activity. Protein–protein interaction are then monitored in living cells following addition of the Nano-Glo Live Cell Reagent, a non-lytic detection reagent containing the cell-permeable furimazine substrate and observed luminescent signals.

To generate different NanoBiT fusion constructs, human ER and PELP1 coding sequences were amplified by PCR and separately subcloned into NB-MCS vectors (Promega). To test the protein–protein interaction between ER and PELP1 by using the NanoBiT assay, C-LgBit-ESR1 paired with C-SmBit-PELP1 or C-LgBit-PELP1 with C-SmBit-ESR1 constructs were transiently transfected into HEK-293T cells by using Fugene HD transfection reagent (Promega). To test the ER dimerization, C-SmBit-ESR1 were cotransfected with either N-LgBit-ESR1 or C-LgBit-ESR1 constructs. On the day after transfection, the medium for the HEK-293T cells was changed to phenol red-free DMEM containing 1% charcoal-stripped FBS. After a 24 hr incubation, the cells were treated with DMSO or ERX-11 (10 μM) for 2 hr and then treated cells with EtOH or E2 (10 nM) for 30 mins. After treatment, Nano-Glo live cell reagents were added into cells and luminscence was measured after 10 mins.