NanoBiT–β-arrestin recruitment assay

For the NanoBiT–β-arrestin recruitment assay (58), a receptor construct was designed to fuse the small fragment (SmBiT) of the NanoBiT complementation luciferase to the C terminus of HA-BLT1 with a 15–amino acid flexible linker (GGSGGGGSGGSSSGG), whose sequences were recommended by the manufacturer (Promega). The construct (HA-BLT1-SmBiT) was assembled and inserted into a pCAGGS mammalian expression plasmid (a gift from J.-i. Miyazaki, Osaka University) using an NEBuilder HiFi DNA Assembly system (New England Biolabs). A β-arrestin construct was generated by fusing the large fragment (LgBiT), whose nucleotide sequences were gene-synthesized with mammalian codon optimization (GenScript), to the N terminus of human β-arrestin1 or β-arrestin2 (βarr1 or βarr2, respectively) with the 15–amino acid linker. The LgBiT-βarr1 and LgBiT-βarr2 were inserted into the pCAGGS plasmid. HEK293A cells (Thermo Fisher Scientific) were seeded in a 10-cm dish at 2 × 10⁶ cells in 10 ml of DMEM, supplemented with 10% FBS and penicillin (100 U/ml) and streptomycin (100 μg/ml), and cultured for 1 day. The cells were transfected with a mixture of HA-BLT1-SmBiT plasmid (1 μg) and the LgBiT-βarr plasmid (LgBiT-βarr1 or LgBiT-βarr2; 500 ng) by diluting in 500 μl of Opti-MEM I Reduced Serum Medium (Thermo Fisher Scientific) and combining 25 μl of polyethylenimine reagent (1 mg/ml) [Polyethylenimine “Max” (molecular weight 40,000), Polysciences] diluted in 500 μl of the Opti-MEM. As a negative control, the pCAGGS plasmid was used. Twenty-four hours after the addition of the transfection solution, the cells were harvested with 5 ml of a 0.53 mM EDTA-containing Dulbecco’s phosphate-buffered saline (D-PBS), followed by rinsing with 5 ml of HBSS containing 5 mM Hepes (pH 7.4). The cells were centrifuged at 190g for 5 min and suspended in 10 ml of 0.01% BSA (fatty acid–free grade; SERVA)–containing HBSS. The cell suspension was seeded in a 96-well white plate at a volume of 80 μl per well and loaded with 20 μl of 50 μM coelenterazine (Carbosynth) diluted in the BSA-HBSS. After incubation at room temperature for 2 hours, background luminescent signals were measured using the luminescent microplate reader SpectraMax L equipped with two detectors (Molecular Devices). Prediluted LTB₄ solution (20 μl at final concentrations of 100 pM to 100 nM, 3.2-fold dilution) or vehicle was manually added to the cells. After ligand addition (5 min), luminescent signals were measured for 5 min with 20-s intervals. For each well, the luminescent signal was normalized to the initial count and fold change values over 5 to 10 min after ligand stimulation was averaged. The fold change βarr recruitment signals were fitted to a four-parameter sigmoidal concentration-response curve, and EC₅₀ (median effective concentration) values were obtained, using the Prism 7 software (GraphPad Prism).