2.5. BRET assay

The BRET assay used for detecting the protein–protein interaction as described previously33. At 48 h post-transfection, cells were washed harvested and adjusted to 1 × 10⁶/mL. An aliquot of 1 μL Coelenterazine-h (Promega, USA) was added into 100 μL cells at a final concentration of 5 μmol/L. The emitted light was determined with a VICTOR X5 Multilabel Plate Reader. Readings at 475 nm (reflecting the bioluminescence given off by RLUC) and 530 nm (reflecting the resonance energy transfer from RLUC to EYFP) were measured simultaneously. The BRET ratio was calculated as emission at 530 nm (light emitted by EYFP)/emission at 475 nm (light emitted by RLUC). The BRET ratios reported were corrected by subtracting the ratios obtained in cells expressing the donor only (RLUC).