BRET measurement
After coelenterazine H (Coel H, Promega, 2.5 µM) addition, ebBRET measurements in cell population were performed using the LB940Mithras plate reader (Berthold) as previously described , on cells seeded at 5 x 104 cells per well in 96-wells white bottom plates and transfected with pcDNA3.1-rGFP-CAAX (33,3 ng/well), pcDNA3.1-p63RhoGEF-RlucII (5,1 ng/well), pcDNA3.1-Gαq (6,75 ng/well) combined with the pRK5-Myc-mGlu5a (14,7 ng/well) or pRK-SNAPtag-AT1 (14,8 ng/well). BRET, or the 535 nm/485 nm ratio, was assessed by calculating the ratio of the light emitted by the acceptor (510–550 nm band-pass filter, Em535) to the light emitted by the donor (460–500 nm band-pass filter, Em480).
Single cell BRET imaging was previously described . Acquisitions were performed on cells seeded in 35mm diameter glass dishes (MatTek® for microscopy) coated with poly-ornithine (0.03 mg/ml, for 30min), after Coel H (5 µM) addition, using the Axio Observer 7 KMAT fluorescence microscope (Carl Zeiss), equipped with a Neofluar 40X/1.3 planar EC oil objective (M27, ZEISS) and an ORCA-Quest qCMOS camera (Hamamatsu), controlled by Metamorph software. The emission of RlucII (10s exposure, FF01-450nm/70-25 Brightline filter, Semrock) and rGFP (20s exposure, HQ535nm/50M filter, Chroma Technology Corp) were measured successively. Images were analyzed using the BRET Analyzer macro on Fiji . Overall, this analysis uses an open-source toolkit for Fiji (https://github.com/ychastagnier/BRET-Analyzer) performing four key steps in the analysis: (1) background subtraction from the image, (2) image alignment over time, (3) composite thresholding of the image, and (4) pixel-by-pixel division of the image and distribution of the BRET ratio intensity on a pseudo-color scale. The BRET ratio corresponds to the acceptor/donor emission ratio (535/450).