Cell culture and transient transfection of HEK293 cells

Human embryonic kidney cells (HEK293) were maintained in a humidified incubator with 5% CO2 and 90% humidity at 37 °C. Cells were grown as a monolayer in Dulbecco’s modified Eagle’s medium (DMEM) with high glucose (25 mM) and pyruvate (1mM) supplemented with 10% (v/v) fetal bovine serum, 100 units/mL penicillin and 0.1 mg/mL streptomycin. Cells were regularly tested for mycoplasma contamination (EZ-PCR™ Mycoplasma Detection Kit, Kibbutz Beit-Haemek, Israel). For experiments, cells were seeded at a density of 2×105to each well of the clear 24-well plate. Before transient transfection cells were grown to 60 to 80% confluence. Transfection was performed using Lipofectamine™ LTX with PLUS™ transfection reagent according to the manufacturer’s instructions. Biosensors were used in transfection as explained in the previous reports [28-30]. Assays were performed 24h after transfection as confluency reaches 80%.

Membrane preparation and Western blot analysis

Cells were washed with ice-cold PBS buffer and lysed with RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% Nonidet P-40 (NP-40), 0.1% SDS, 0.5% sodium deoxycholate, 1mM EDTA, 1 mM DTT, 0.5 mM PMSF, 50 mM Na β-glycerophosphate, 1 mM NaF and 1× Protease inhibitor cocktail tablet). Extracts were centrifuged for 30 minutes at 16,000×g at 4°C. Protein concentration was measured with Pierce™ BCA (Bicinchoninic acid) protein assay kit. Membrane proteins were solubilized in SDS sample buffer and 20 μg of total protein from cells expressing GPR56 constructs were resolved by SDS-PAGE and transferred to Immobilon-P PVDF membrane (Millipore Corporation, USA) Blotting membranes were blocked with 5% non-fat dry milk in TBST for 1 hour at room temperature and followed by 3 washes with TBST. Blotting membranes were incubated with 1:500 diluted anti-GPR56 or anti-β-actin primary antibodies overnight at 4°C. Next day, blotting membranes were washed 3 times with TBST and incubated with 1:7500 diluted HRP conjugated secondary antibody for 1 hour at room temperature. After the secondary antibody incubation, blotting membranes were washed with TBST and briefly incubated with SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Pierce, USA) according to the manufacturer’s instructions. Blots were imaged using ChemiDocTM MP imaging system (BioRad, USA).

12, Gα13, Gα11 BRET biosensor assay

For Gα BRET biosensor assay, HEK293 cells were washed with BRET buffer [29] and gently detached from 24-well plate on the day of the assay. Cells were centrifuged at 900 rpm for 5 minutes and 7.5×104 HEK293 cells were seeded into each well of a white opaque 96-well microplate. Triplicate wells were loaded with P7Stachel peptide (TYFAVLM ) with a final concentration of 1mM and 1:1000 ratio of Furimazine. After 5 minutes of incubation in the dark, bioluminescence and fluorescence signals were measured using Berthold Mithras2 LB 943 multimode plate reader using MicroWin 2010 software (Berthold Technologies, Germany) equipped with high-efficiency BRET filters. For Nluc emission, NanoBRET filter (460nm/70) and for Venus emission, eYFP (540nm/40) filters were used in biosensor-based BRET experiments. Data acquired represents at least from three independent experiments done in triplicates. The statistical evaluation of quiescent receptor vs. Stachel stimulation within each construct was done by multiple t test. The comparison of eachBFPP mutant with the wild-type GPR56 was done using ordinary one-way ANOVA and Dunnett’s multiple comparison tests.

β-Arrestin BRET biosensor assay

For β-arrestin recruitment BRET biosensor assay, HEK293 cells were washed with BRET buffer and gently detached from 24-well plate on the day of the assay. Cells were centrifuged at 900rpm for 5 minutes and 7.5×104 HEK293 cells were seeded into each well of a white opaque 96-well microplate. Wells were loaded with P7Stachel peptide (TYFAVLM ) to give a final concentration of 1 mM and with Coelenterazine native to yield a final concentration of 5 μM per well. After 5 minutes of incubation in the dark, bioluminescence and fluorescence signals were measured using Berthold Mithras2 LB 943 multimode plate reader using MicroWin 2010 software (Berthold Technologies, Germany) equipped with high-efficiency BRET filters. For Citrine emission, eYFP filter (540nm/40) and for RLuc8 emission, Coelenterazine filter (480nm/20) were used in experiments. Data acquired represents at least from three independent experiments done in triplicates. The statistical evaluation of quiescent receptor vs. Stachel stimulation within each construct was done by multiple t test. The comparison of eachBFPP mutant with the wild-type GPR56 was done using ordinary one-way ANOVA and Dunnett’s multiple comparison tests.