Bioluminescence resonance energy transfer (BRET) assay

For BRET assay, HEK-293T cells were transiently cotransfected with a constant amount of cDNA encoding for GluN1-RLuc (0.25 μg) and GluN2B (0.15 μg) and with increasing amounts of cDNA corresponding to CB₂R-YFP (0.25 to 1.25 μg). As a negative control, HEK-293T cells were transiently cotransfected with a constant amount of cDNA encoding for GluN1-RLuc (0.25 μg) and GluN2B (0.15 μg) and with increasing amounts of cDNA corresponding to GHSR1a-YFP (0.25 to 1.5 μg). To control the cell number, sample protein concentration was determined using a Bradford assay kit (Bio-Rad, Munich, Germany) using bovine serum albumin (BSA) dilutions as standards. To quantify fluorescent proteins, cells (20 μg of total protein) were distributed in 96-well microplates (black plates with a transparent bottom) and fluorescence was read in a Fluostar Optima Fluorimeter (BMG Labtech, Offenburg, Germany) equipped with a high-energy xenon flash lamp, using a 10-nm bandwidth excitation filter at 485 nm. For BRET measurements, the equivalent of 20 μg of total protein cell suspension was distributed in 96-well white microplates with a white bottom (Corning 3600, Corning, NY). BRET was determined one minute after adding coelenterazine H (Molecular Probes, Eugene, OR), using a Mithras LB 940 plate reader (Berthold Technologies, DLReady, Germany), which allows the integration of the signals detected in the short-wavelength filter at 485 nm and the long-wavelength filter at 530 nm. To quantify GluN1-RLuc expression, luminescence readings were obtained 10 min after the addition of 5 μM coelenterazine H. MilliBRET units (mBU) are defined as:

where C_f corresponds to [(long-wavelength emission)/(short-wavelength emission)] for the RLuc construct expressed alone in the same experiment.