Furimazine

Real-time BLI was performed using an In Vivo Imaging System (IVIS) Spectrum imager (PerkinElmer, USA) following the administration of furimazine—Nano-Glo Luciferase Assay substrate (Promega #N1120). For all in vitro BLI, cells were treated with 50 µM furimazine in Nano-Glo Luciferase Assay Buffer (Promega #N1120) for 5 min according to the manufacturer’s protocol. Then Bioluminescent images were captured with an open filter, binning set to 4. For in vivo BLI, animals were anesthetized with isoflurane prior to the subcutaneous injection of 250 µM furimazine (Promega #N1120; 1/20 dilution) into the calvaria defect site. Images were captured with an open filter, binning set to 4, and acquisition times of 60 s at the indicated settings. All BLI signal detected (both in vitro and in vivo) using the GpNLuc reporter represent BRET signal deriving from intramolecular energy transfer between NanoLuc and eGFP. Total flux (p/s) and average radiance (p/s/cm²/sr) were calculated using the Living Image software (PerkinElmer, USA).

For whole cell NanoBRET saturation assays, HEK293T cells were transfected with secNluc-D₃R employing Mirus TransIT-293 (3:1 reagent to DNA ratio). After 24 h at 37 °C, 5% CO₂, cells were detached using Versene and transferred to white, F-bottom 384-well plates at a density of 2,500 cells/well and incubated for another 24 h at 37 °C, 5% CO₂. Cells were washed with PBS and incubated for 30 min at 37 °C with 5% FBS in DMEM without phenol red, before the fluorescent ligands (5 µL, diluted into DMEM without phenol red with 5% FBS from a 1 mM DMSO-stock) were added. Non-specific binding was determined in the presence of 10 µM haloperidol. After 1 h incubation with the ligands at 37 °C, furimazine (Promega, final dilution 1:500 to 1:2,500) was added, followed by a 5 min incubation at room temperature in the dark. BRET was measured as the ratio of acceptor fluorescence and donor luminescence employing a CLARIOstar microplate reader equipped with 475/30 nm and 535/30 or 580/30 emission filters, respectively. Total, non-specific and specific binding were analyzed employing the algorithms for one-site saturation binding implemented in PRISM6.0 or 8.0.

Epidermal Growth Factor fluorescently labelled with Alexa Fluor 488 (E13345) or Alexa Fluor 647 (E35351) were purchased from Thermo Fischer Scientific (Waltham, USA). Human recombinant TGF-alpha (239-A-100), human recombinant betacellulin (261-CE-010), human recombinant epiregulin (1195-EP-025), human recombinant amphiregulin (262-AR-100), human recombinant epigen (6629-EP-025) and human recombinant EGF (236-EG-200) were purchased from R&D Systems (Minnesota, USA). Purified LgBiT, FuGENE HD Transfection Reagent and furimazine were purchased from Promega Corporation. Opti-MEM reduced serum medium was purchased from Gibco (31985062). Q44c and Q86c, containing an unpaired cysteine in the C-terminal tag, were provided by QVQ (Utrecht, The Netherlands).

Essentially as described, the LIPS assays were performed in approximately 2.5 h using a 96-well plate format at room temperature [19 (link),20 (link),21 (link)]. Briefly, recombinant luciferase antigen lysates were produced from transfection of DNA plasmids constructs into Cos1 cells and their activity in light units (LU) was determined with a tube luminometer (Turner Design 20/20). To initiate testing, 40 μL of buffer A, 10 μL of diluted human sera (1 μL equivalent), and the luciferase-antigen cell extract (input of approximately 10 million LU), diluted in buffer A, were added to each well of a microtiter plate for 1 h. Next, a 30% suspension (6 μL) of Ultralink protein A/G beads (Pierce Biotechnology, Rockford, IL, USA) was added to the bottom of each well of a 96-well filter HTS plate (Millipore, Bedford, MA, USA). The 100 μL antigen-antibody reaction mixture was moved to a filter plate and incubated for 1 h on a rotary shaker. Multiple washing steps of the retained protein A/G beads were then performed. After completion of the washing steps, LU were measured in a Berthold LB 960 Centro microplate luminometer (Berthold Technologies, Bad Wildbad, Germany) using either coelenterazine or furimazine substrate (Promega, Madison, WI, USA) for detecting Renilla luciferase and NanoLuc activity, respectively.

D-Ala²,N-Me-Phe⁴,Gly⁵-ol-enkephalin was obtained from Mimotopes. Morphine, Rhodamine 6G and M2-anti-FLAG were from Sigma-Aldrich (Gillingham, Dorset, United Kingdom). Naloxone was from Tocris. SNAP-Surface 488 was from New England Biolabs (Ipswich, MA, United States). The antibody anti-pS375 was from Cell Signaling. Secondary antibodies (raised in donkey) conjugated to Alexa-Fluor 488 or 647 were from Jackson ImmunoResearch. Coelenterazine h was from NanoLight. Furimazine was from Promega. Compound 101 was from HelloBio. Pitstop2 was from Abcam. PTx was from Millipore.