1 to 80 mg of CRA13

CHO-CB2 cells were plated overnight onto poly-D-lysine pre-coated 24 well plates (Corning - cat no. 3667) at a density of 2 x 10^5 cells per well. For the experiment, cells were washed once with 500 μl stop-medium (DMEM without FBS) and a further 250 μl stop medium containing 3 μCi/ml 2-[3H]adenine added to the cells and incubated for 3 h at 37°C. The media was aspirated and the cells washed 2 x with 500 μl HBS buffer (130 mM NaCl, 0.9 mM NaH2PO4, 0.8 mM MgSO4, 1.8 mM CaCl2, 5.4 mM KCl, 25 mM glucose, 20 mM HEPES, pH 7.4 with phenol red added at 5 mg/l) with a further 500 μl HBS added. Where required IBMX (1 mM final concentration), forskolin (10 μM final concentration) and compound (CB-13, at varying concentrations from 100 x stock solutions in DMSO) were added and the plates incubated for 15 min in a water bath at 37°C. The media was aspirated and 500 μl cold 5% (v/v) TCA added to stop the reaction. A further 500 μl cold 5% (v/v) TCA which contained 1 mM ATP and 1 mM cAMP (to block non-specific binding sites) was added and the plate incubated at 4 °C for 20 min. The resultant extract was loaded directly onto pre-equilibrated dowex columns. A further 2.5 ml H2O was added and ATP drained from the columns. The dowex columns were placed directly above preequilibrated alumina columns and 8 ml H2O added and allowed to flow through both columns. Scintillation vials were placed beneath the alumina columns and 6 ml 0.1 M imidazole added to elute the bound cAMP. 10 ml of scintillation fluid (IREA-Safe, Perkin-Elmer) was added to each vial, lids applied, the vials shaken and counted on a Wallac 1409 Liquid Scintillation Counter for 2 min, with dpm’s reported. Nucleotide binding and cAMP data were analyzed by non-linear regression with the software Origin, version 7.5, from Origin Lab Corporation, MA, US.