Filtermat b

The membrane suspension, ³H-histamine (PerkinElmer, USA), and cold ligand (histamine) were diluted with the assay buffer (20 mM HEPES pH7.5, 150 mM NaCl). Briefly, 100 μL of the reaction mixture was dispensed in triplicate in a 96-well microplate. Membrane proteins (300 μg) were added to each well. The final concentrations of ³H-histamine were 200, 100, 50, 25, 12.5, 6.25, and 3.13 nM. To achieve non-specific binding, the experiment was performed in the presence of a cold ligand at a 1000-fold concentration of the hot ligand. The reaction mixture was incubated at room temperature (22–24 °C) for 1 h. The membrane was harvested on a glass fiber Filtermat B (PerkinElmer, USA) filter paper presoaked in 0.3% polyethylene imine using a FilterMate cell harvester (PerkinElmer, USA). The filter paper was washed with distilled water and dried. The solid scintillator, MeltiLex B/HS (PerkinElmer, USA), was melted on the filter paper. Radioactivity was detected using a Microbeta 2 system (PerkinElmer, USA). Specific binding was determined by subtracting nonspecific binding from total binding.

^[35]S-labeled methionine and ^[35]S-labeled cysteine incorporation in nascent proteins was measured according to the manufacturer’s instruction (EasyTag EXPRESS³⁵S Protein Labeling Mix, Perkin Elmer, Upplands Väsby, Sweden). Briefly, 10⁵ cells were seeded per well in six well plates, allowed to attach overnight and treated in methionine and cysteine free DMEM (Gibco Thermo Fisher) with RITA in presence or absence of ISRIB at indicated concentrations for 4 h. Next, cells were incubated for 30 min in DMEM supplemented with S35 labeled Met and Cys (20 µCi/ml), after which they were washed three times with PBS and lysed with 100 µl radio-immunoprecipitation assay buffer (RIPA buffer; 100 mM sodium chloride, 1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris pH 8.0 [Sigma-Aldrich]). The lysate was centrifuged for 10 min at 20.000 rpm in a tabletop centrifuge and 15 µl of the supernatant was spotted on a glass fiber filtermat (Filtermat B, Perkin-Elmer). The filtermat was subsequently washed twice in 10% Trichloroacetic acid (TCA) and once with ethanol:acetone (50:50) for 10 min each and dried overnight. A melt-on scintillator (MeltiLex, Perkin-Elmer) was applied to the filtermat and counts per minute were monitored using a microBeta plate reader (MicroBeta2, Perkin Elmer).

Two micrograms of human GhrR membranes (prepared from HEK293 cells stably expressing the fusion protein hGhrR-eYFP), 60 pM [¹²⁵I-His⁹]ghrelin (PerkinElmer, Waltham, MA, USA), and different concentrations of the test compound were incubated in HEPES buffer (20 mM HEPES, 20 mM CaCl₂, 0.8 mM MgCl₂, 1% Pefabloc, 1% bovine serum albumin, pH 7.4) in a 96-well plate on a shaker (200 rpm) for 2.5 h at rt. Each concentration was tested in duplicate. The membrane-bound radioligand was trapped on a Filtermat B (PerkinElmer) pre-soaked with 0.1% polyethylenimine in H₂O by using a filtermat harvester (PerkinElmer). After drying for 20 min at 55 °C, a Meltilex scintillation sheet (PerkinElmer) was melted onto the filtermat and the samples were counted in a Microbeta scintillation counter (PerkinElmer). For each compound, all data points (n = 4–6) from at least two independent experiments were combined and IC₅₀ values were obtained by a sigmoidal dose-response fit. Data were normalized to no competition (100%, minimal ghrelin effect) and full competition (0%, maximal ghrelin effect).