Radioligand displacement
experiments were performed as in previously published methods.7 (link) Membrane aliquots containing 15 μg of protein
were incubated in a total volume of 100 μL assay buffer (50
mM Tris–HCl, 5 mM MgCl2, supplemented with 0.01%
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and 1 mM
ethylenediaminetetraacetic acid (EDTA), pH 7.4) at 25 °C for
120 min. Displacement experiments were performed using six concentrations
of competing antagonist in the presence of ∼10 nM [3H]PSB-11. Nonspecific binding was determined in the presence of 100
μm NECA and represented less than 10% of total binding. Incubation
was terminated by rapid filtration performed on 96-well GF/B filter
plates (PerkinElmer, Groningen, the Netherlands) in a PerkinElmer
Filtermate-harvester (PerkinElmer, Groningen, the Netherlands). After
the filter plate was dried at 55 °C for 30 min, the filter-bound
radioactivity was determined by scintillation spectrometry using a
2450 MicroBeta2 Plate Counter (PerkinElmer, Boston, MA).
experiments were performed as in previously published methods.7 (link) Membrane aliquots containing 15 μg of protein
were incubated in a total volume of 100 μL assay buffer (50
mM Tris–HCl, 5 mM MgCl2, supplemented with 0.01%
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and 1 mM
ethylenediaminetetraacetic acid (EDTA), pH 7.4) at 25 °C for
120 min. Displacement experiments were performed using six concentrations
of competing antagonist in the presence of ∼10 nM [3H]PSB-11. Nonspecific binding was determined in the presence of 100
μm NECA and represented less than 10% of total binding. Incubation
was terminated by rapid filtration performed on 96-well GF/B filter
plates (PerkinElmer, Groningen, the Netherlands) in a PerkinElmer
Filtermate-harvester (PerkinElmer, Groningen, the Netherlands). After
the filter plate was dried at 55 °C for 30 min, the filter-bound
radioactivity was determined by scintillation spectrometry using a
2450 MicroBeta2 Plate Counter (PerkinElmer, Boston, MA).
