CHEMBL3887273

Competition Binding Assay: Competition binding experiments have been performed incubating membranes from CHO-K1 cells stably transfected with the human adenosine A1 receptor (cat. ES-010-M400UA, Perkin Elmer, Boston, Mass., USA) (5-10 ug of protein/sample) with a single concentration of [3H]DPCPX (1.7 nmol/l) (Perkin Elmer), in the presence of various concentrations (ranging from 10E-5 to 10E-10 M) of cold DPCPX, ST4206 and ST4208 in 96-well filter plates (MultiScreen system, cat #MAFBN0B10, Millipore, Billerica, Mass., USA) for 60 min at 25 C. in a total volume of 200 uL/well of 25 mmol/l Hepes, 5 mmol/L MgCl2, 1 mmol/l CaCl2, 100 mmol/L NaCl, pH 7.4 (all from Sigma-Aldrich). Non-specific binding has been determined in the presence of 250 umol/l of cold DPCPX (8-cyclopentyl-1,3-dipropylxanthine, Sigma-Aldrich). At the end of incubation, bound and free radioligands have been separated by filtering the 96-well filter plates using a Millipore filtration apparatus.