Nanoluc substrate furimazine

propranolol‐Peg8‐BY630 (propranolol‐peg8‐BODIPY630/650) and prop‐β(Ala‐Ala)‐BY630 (propranolol‐βalanine‐ βalanine‐X‐BODIPY630/650) were obtained from CellAura (Nottingham, UK). BODIPY‐TMR‐CGP (CGP‐12177‐TMR) was purchased from Molecular Probes (Eugene, OR). propranolol, ICI 118551, CGP 12177, CGP 20712A, and cimaterol were from Tocris (Bristol, UK). Isoprenaline was purchased from Sigma‐Aldrich (Gillingham, UK). The NanoLuc substrate furimazine was obtained from Promega (Southampton, UK). The radioligand ³H CGP 12177 was obtained from PerkinElmer (Coventry, UK).

Saturation‐ and competition‐binding assays were performed on stably transfected HEK 293 cells. Cells were seeded 24 h before experimentation in white Thermo Scientific 96‐well microplates. The medium was removed from each well and replaced with HEPES‐buffered saline solution (HBSS; 147 mmol/L NaCL, 24 mmol/L KCl, 1.3 mmol/L CaCl₂, 1 mmol/L MgSO₄, 10 mmol/L HEPES, 2 mmol/L sodium pyruvate, 1.43 mmol/L NaHCO₃, 4.5 mmol/L d‐glucose, pH 7.2–7.45) with the relevant concentration of fluorescent ligand and, if necessary, competing ligand. Nonspecific binding was determined with 10 μmol/L propranolol. Cells were then incubated in the dark for 1 or 2 h at 37°C (no CO₂). The NanoLuc substrate furimazine (Promega) was then added to each well at a final concentration of 10 μmol/L and allowed to equilibrate for 5 min prior to reading. We measured the luminescence signals at two different wavelengths using the PHERAstar FS plate reader (BMG Labtech, UK) at room temperature. The filtered light from each well was simultaneously measured using 460 nm (80‐nm bandpass) and >610 nm longpass filters. The resulting raw BRET ratio was calculated by dividing the >610 nm emission by the 460 nm emission.

Cells were first treated with olmesartan medoxomil, LVV-H7 and/or vehicle (as described in figure legends) for 20 min at 37 °C followed by addition of increasing doses of BODIPY-AngII and incubation for a further 40 min at 37 °C. The NanoLuc substrate furimazine (Promega) was then added to a final concentration of 10 μM and light emission was measured at 37 °C using the filters 450 nm (80-nm bandpass) and >610 nm (longpass) on the LUMIstar or CLARIOstar multilabel plate reader (BMG Labtech (Australia), Mornington, VIC, Australia).