Isoplate 96 plates

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 receptors, 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-PACAP27 (40 pM, PerkinElmer) and seven decreasing concentrations of unlabeled peptides were added separately 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 per minute, CPM) in a scintillation counter (MicroBeta2 Plate Counter, PerkinElmer) using a scintillation cocktail (OptiPhase SuperMix, PerkinElmer). The span data were normalized to the WT (span of WT was defined as 100%).

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.

HEK293 cells were cultured in DMEM 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 WT or mutant constructs, cells were washed twice and incubated with blocking buffer (DMEM 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]-[Nle⁴,d-Phe⁷]-α-MSH (30 pM, PerkinElmer) and seven decreasing concentrations of unlabeled peptide (γ-MSH (50 μM to 128 pM) or α-MSH (1 μM to 4 pM) or PG-901 (1 μM to 4 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 per minute, CPM) in a scintillation counter (MicroBeta² Plate Counter, PerkinElmer) using a scintillation cocktail (OptiPhaseSuperMix, PerkinElmer).