Radioligand Binding桯EK293 cells (4 x 106 cells/100-mm dish) were transfected with 0.5 礸 of S138R or wild-type 5-HT2C receptor plasmid DNA using 20 祃 of Lipofectamine. For saturation experiments, HEK293 and S138R-expressing stable cells were transfected with 0.2 or 0.5 礸 of wild-type 5-HT2C receptor plasmid DNA. Membranes were prepared from transfected cells as described previously (23). Saturation experiments were performed using [3H]mesulergine (0.1-5 nM) in the presence and absence of 1 ?FONT size=-2>M mianserin in 0.5 ml of buffer (50 mM Tris-HCl, 10 mM MgSO4, 0.5 mM EDTA, and 0.1% ascorbate) with 0.5 ml of diluted cell membranes (10 礸 of protein/assay tube). The mixture was incubated at 37 癈 for 30 min, followed by filtration through glass fiber filters (Schleicher & Sch黮l) on a Brandel cell harvester and liquid scintillation counting in Ecoscint (National Diagnostics). Protein was determined using BCA (Pierce). Data were analyzed using GraphPad Prism software.
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