Furimazine nanoglo

Coelenterazine was purchased from Carbosynth. Linear polyethylenimine (PEI, 25 kDa) was supplied by Polyscience. FuGENE® HD Transfection Reagent, furimazine (NanoGlo®), HaloTag® NanoBRET™ 618 ligand and T4 DNA ligase were from Promega. Phosphosite-specific antibodies pS355/pS356-β2 (Cat. no.: 7TM0029A) and pT360/pS364-β2 (Cat. no.: 7TM0029B) were from 7TM Antibodies, and the antibody against pS261 (Cat. no.: PA5-12977) was from Invitrogen. Anti-HA-tag antibodies were purchased from Cell Signaling Technology. Hanks balanced salt solution (HBSS), Dulbecco’s modified Eagles’s medium (DMEM), Lipofectamin 2000, HA-beads, TetraSpeck fluorescent beads, 96-well white polystyrene LumiNunc microplates, all antibiotics and pertussis toxin (PTX) were from Thermo Fisher. The HTRF cAMP accumulation kit and the DERET substrate, SNAP-Lumi4-Tb, were purchased from Cisbio. SNAP-Surface Alexa Flour 488, SNAP-Surface Alexa Flour 549, SNAP-Surface Alexa Flour 649, Q5® High-Fidelity polymerase, NotI-HF®, ApaI, anti-SNAP-tag antibody (Cat. no.: P9310S) were obtained from New England Biolabs (NEB). Effectene was from Qiagen. All other chemicals and compounds were from Sigma Aldrich. TopSeal-A PLUS, Dihydroalprenolol Hydrochloride ([³H]-DHA, 250µCi, 9.25MBq), WGA PVT 500 MG SPA Beads, and OptiPlate-96, and White Opaque 96-well Microplates were from Perkin Elmer.

Cells were washed with DPBS, harvested by trituration, and transferred to opaque black or white 96-well plates containing diluted drug solutions. For assays with nucleotide-free heterotrimers (Fig. 4), cells were washed with permeabilization buffer containing 140 mm KCl, 10 mm NaCl, 1 mm MgCl₂, 0.1 mm K-EGTA, 20 mm NaHEPES (pH 7.2), harvested by trituration, and permeabilized in the same buffer containing 10 μg ml⁻¹ high purity digitonin (EMD Millipore, Burlington, MA). Measurements were made from permeabilized cells supplemented either with GDP (0.5 mm) or apyrase (2 units ml⁻¹; Sigma) and agonist. BRET and luminescence measurements were made using a Mithras LB940 photon-counting plate reader (Berthold Technologies GmbH, Bad Wildbad, Germany). Coelenterazine h (5 μm; Nanolight, Pinetop, AZ) or furimazine (Nano-Glo; 1:1000, Promega Corp.) were added to all wells immediately prior to making measurements with Rluc8 and Nluc (or Nluc fragments), respectively. Raw BRET signals were calculated as the emission intensity at 520–545 nm divided by the emission intensity at 475–495 nm. Net BRET was this ratio minus the same ratio measured from cells expressing only the BRET donor. NES–venus–mG fluorescence in Fig. 2 was measured using a Guava 6HT/2L flow cytometer (excitation 488 nm, detection 525/30 nm) and reported as average fluorescence from all positive cells.

HEK 293 cells transiently transfected with WT or mutant 5-HT₇ receptor plasmids plus either the Nluc-EPAC-VV cAMP sensor (at a 15:1 ratio) or Venus-kras (at a 1:8 ratio) (Tian et al., 2017 (link)) were incubated for 24 hr. After washing twice with PBS, they were transferred to opaque black 96-well plates. Steady-state BRET measurements were made using a Mithras LB940 photon-counting plate reader (Berthold Technologies GmbH, Bad Wildbad, Germany). Furimazine (NanoGlo; 1:1000, Promega) for cAMP measurement or coelenterazine h (5 µM; Nanolight, Pinetop, AZ) for trafficking assay was added followed by BRET signal detection and calculation at an emission intensity of 520–545 nm divided by that of 475–495 nm.