D4 Dopamine Receptor radioligand binding assay.

Binding was measured using HEK293T membrane preparations transiently expressing human D₂ (D₂ long receptor), D₃, and D₄ (D_4.4 variant). HEK293T cells (ATCC CRL-11268; 59587035; mycoplasma free) were transfected and membrane preparation and radioligand binding assays were set up in 96-well plates in the standard binding buffer (50 mM Tris, 10 mM MgCl₂, 0.1 mM EDTA, 0.1% BSA, pH 7.4)¹⁴. For primary screening, 10 μM compounds were incubated with membrane and radioligands (0.8–1.0 nM [³H]-N-methylspiperone) (PerkinElmer). For displacement experiments, test compounds with increasing concentrations were incubated with membrane and radioligands (0.8–1.0 nM [³H]-N-methylspiperone). Reactions, either primary screening or displacement experiments, were incubated for 2 h at room temperature in the dark and terminated by rapid vacuum filtration onto chilled 0.3% PEI-soaked GF/A filters followed by three quick washes with cold washing buffer (50 mM Tris HCl, pH 7.4) and quantified as described previously⁷³. Results (with or without normalization) were analyzed using GraphPad Prism 5.0 using one-site shift models where indicated.