BRET measurements

One day after transfection, HEK293 cells were detached and seeded onto polyornithine-coated white 96-well plates at a density of 2.5 × 10⁴ cells per well in media. The next day, cells were washed once with Tyrode’s buffer [140 mM NaCl, 2.7 mM KCl, 1 mM CaCl₂, 12 mM NaHCO₃, 5.6 mM d-glucose, 0.5 mM MgCl₂, 0.37 mM NaH₂PO₄, 25 mM Hepes (pH 7.4)] and left in Tyrode’s buffer. For kinetic measurements, BRET signals were monitored every 2 s after addition of the cell-permeable coelenterazine 400a at a final concentration of 5 μM, using a Synergy2 (BioTek) microplate reader. The filter set was 410/80 nm and 515/30 nm for detecting the RlucII (Renilla luciferase) (donor) and GFP10 (acceptor) light emissions, respectively. AngII (at a final concentration of 100 nM) was injected after 11 measurements (20 s), and BRET was recorded over a period of 5 min. For concentration-response curves of the Gα_q-poly, Gα_i2 and Gα_i3, p63/Gα_q, and Rho sensors, BRET signals were measured after the cells were stimulated with various concentrations of ligand in Tyrode’s buffer for 2 min at room temperature (21°C). For detecting DAG, PKC-c1b, and Lyn-PKC sensors, cells were stimulated with ligands for 1 min before BRET measurement. For the Gα₁₂ sensor, cells were stimulated for 10 min at 37°C. For detecting βarr2 at the PM or in endosomes, cells were stimulated with either various concentrations of AngII for 4 min at room temperature or for 30 min at 37°C, before BRET measurements. Coelenterazine 400a (final concentrations of 5 μM) was added 3 to 5 min before BRET measurements. For the βarr2 sensor, BRET signals were measured 20 min after addition of various concentrations of ligands, and coelenterazine 400a was added 10 min before BRET measurements. The Gα_q/11 inhibitors [UBO-QIC (100 nM) and YM-254890 (200 nM)] and PKC inhibitors [Gö6983 (2 μM), Gö6976 (3 μM), and LY333531 (3 μM)] were added for 30 min before ligand stimulation. PMA and forskolin were used at a concentration of 1 and 10 μM, respectively, for 10 min. A23187 was used at a concentration of 1 μM for 1 min. To inhibit Gα_i, cells were incubated overnight with PTX (100 ng/ml) before ligand stimulation. PD 123319 and losartan were added at a concentration of 10 μM for 30 min, before adding 1 μM AngII. For single-concentration stimulations of the p63/Gα_q sensor, cells were stimulated for 1 min with either AngII (100 nM), PGF2α (100 nM), dopamine (1 μM), or isoproterenol (1 μM). The BRET ratio was determined by calculating the ratio of the light emitted by GFP10 over the light emitted by the RlucII. All BRET experiments were performed in triplicate.