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).