Microbeta2 link plate counter

This assay was conducted in 96-well Iso-plates (PerkinElmer) coated with poly-D-lysine hydrobromide (Sigma-Aldrich). Twenty-four hour after transfection with GHRHR(23-423) or GHRHR(23-405)-LgBiT-2MBP, HEK 293 T cells were washed twice, and incubated with blocking buffer (DMEM medium supplemented with 33 mM HEPES, and 0.1% (w/v) BSA, pH 7.4) for 2 h at 37 °C. Then, radiolabeled ¹²⁵I-GHRH (80 pM, Phoenix Biotech) and seven decreasing concentrations of unlabeled peptide (10 μM, five serial gradient dilutions) were added and competitively reacted with the cells in binding buffer (PBS supplemented with 10% (w/v) BSA, pH 7.4) at RT for 3 h. Cells were washed with ice-cold PBS and lysed by 50 μL lysis buffer (PBS supplemented with 20 mM Tris-HCl and 1% (v/v) Triton X-100, pH 7.4). Finally, 150 μL of scintillation cocktail (OptiPhase SuperMix, PerkinElmer) was employed and radioactivity (counts per minute) read in a scintillation counter (MicroBeta^{2 (link)} plate counter, PerkinElmer).

For SSTR2 and SSTR4, CHO-K1 cells were cultured in F12 medium with 10% FBS and seeded at a density of 30,000 cells/well in Isoplate-96 plates (PerkinElmer). Twenty-four hours after transfection with the WT or mutant SSTR2/SSTR4, CHO-K1 cells were washed twice and incubated with blocking buffer (F12 supplemented with 25 mM HEPES and 0.1% (w/v) BSA, pH 7.4) for 2 h at 37 °C. For homogeneous competition binding, radiolabeled ¹²⁵I-(Tyr¹¹ ) SST (PerkinElmer; SSTR2, 60 pM; SSTR4, 40 pM) and unlabeled peptide at seven decreasing concentrations (SST-14, 10 μM to 10 pM; L-0545,22, 10 μM to 85 pM; octreotide, 10 μM to 85 pM; CYN 154806, 10 μM to 85 pM; J-2156, 10 μM to 10 pM; peptide 3, 10 μM to 10 pM) were added and competitively reacted with the cells in blocking buffer at RT for 3 h. Following incubation, cells were washed three times with ice-cold PBS and lysed by 50 μL lysis buffer (PBS supplemented with 20 mM Tris-HCl, 1% Triton X-100, pH 7.4). The radioactivity was subsequently counted (counts/min, CPM) in a scintillation counter (MicroBeta^{2 (link)} Plate Counter, PerkinElmer) using a scintillation cocktail (OptiPhaseSuperMix, PerkinElmer). Data were analyzed by nonlinear regression using GraphPad PRISM 8.

The WT or mutant CCK_ARs were transiently transfected into HEK 293T/17 cells (purchased from the Cell Bank at the Chinese Academy of Sciences), which were cultured in a poly-d-lysine-coated 96-well plate. After 24 h, the cells were washed twice and incubated with blocking buffer (Dulbecco’s modified Eagle medium (DMEM) supplemented with 33 mM HEPES, and 0.1% (wt/vol) bovine serum albumin (BSA), pH 7.4) for 2 h at 37 °C. After three washes with ice-cold phosphate-buffered saline (PBS), the cells were treated by a constant concentration of ¹²⁵I-CCK-8 (40 pM, PerkinElmer) plus eight different doses of CCK-8 (1 pM to 10 μM) for 3 h at r.t. Cells were washed three times with ice-cold PBS and lysed by 50 μl of lysis buffer (PBS supplemented with 20 mM Tris-HCl and 1% (vol/vol) Triton X-100, pH 7.4). Subsequently, the plates were counted for radioactivity (counts per minute) in a scintillation counter (MicroBeta^{2 (link)} plate counter, PerkinElmer) using 150 μl of scintillation cocktail (OptiPhase SuperMix, PerkinElmer).