Resonance Energy Transfer

For bioluminescence resonance energy transfer (BRET), HEK-293T cells were transiently cotransfected with a constant amount of cDNA encoding for proteins fused to Rluc and increasing amounts of cDNAs corresponding to proteins fused to YFP (see figure legends). To normalize the number of cells, protein concentration was determined using a Bradford assay kit (Bio-Rad, Munich, Germany) using BSA dilutions as standards. To quantify protein YFP expression, cells (20 μg of protein) were distributed in 96-well plates (black plates with a transparent bottom), and fluorescence was read in the FluoStar Optima Fluorimeter (BMG Labtech, Offenburg, Germany) equipped with a high-energy xenon flash lamp, using a 10-nm bandwidth excitation filter at 400 nm reading. Protein fluorescence expression was determined as fluorescence of the sample minus the fluorescence of cells expressing the BRET donor alone. For BRET measurements, the equivalent of 20 μg of cell suspension was distributed in 96-well plates (Corning 3600, white plates; Sigma), and coelenterazine H (5 μM; Invitrogen) was added. After 1 min, readings were obtained using a Mithras LB 940 (Berthold Technologies), 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 protein-Rluc luminescence, readings were also performed 10 min after addition of coelenterazine H. For SRET assays, cells were transiently cotransfected with constant amounts of cDNA encoding for both receptor fused to Rluc and YFP proteins, and with increasing amounts of cDNA corresponding to the receptor fused to cherry protein. After 48 h of transfection, quantification was performed in parallel in aliquots of transfected cells (20 μg of protein): quantification of receptor YFP or receptor Rluc expression was performed as indicated for BRET experiments. Quantification of receptor-Cherry expression, cells were distributed in 96-well plates (Corning black plates with a transparent bottom), and fluorescence was read in the FluoStar Optima Fluorimeter using a 10 nm bandwidth excitation filter at 590 nm reading. For SRET quantification, cells were distributed in 96-well plates (black plates with transparent bottom), and coelenterazine H (5 μM) was added. After 1 min, the readings were collected using a FluoStar Optima Fluorimeter, which allows the integration of the signals detected in the short-wavelength filter at 530 nm and the long-wavelength filter at 590 nm. Net BRET and net SRET were defined as [(long-wavelength emission)/(short-wavelength emission)] – C_f, where C_f corresponds to [(long-wavelength emission)/(short-wavelength emission)] for the Rluc construct expressed alone in the same experiment. Both fluorescence and luminescence were measured before every experiment to confirm similar donor expressions (∼100,000 bioluminescence units) while monitoring the increase in acceptor expression (1,000–40,000 fluorescence units). BRET or SRET was expressed as, respectively, milliBRET (mBU) or milliSRET (mSU) units (net BRET or SRET × 1,000). Data were fitted to a nonlinear regression equation, assuming a single-phase saturation curve with GraphPad Prism software (GraphPad Software). The relative amount of BRET or SRET is given as a function of 100× the ratio between the fluorescence of the acceptor (YFP or cherry) and the luciferase activity of the donor (Rluc).