mg) in assay buffer B (assay buffer A plus 1.25 g/L BSA) with 0.1–60 μM of anandamide plus URB597 (10 nM) in the presence of 30 μM GDP and 0.1 nM [35S]GTPγS in 0.5 mL total volume for 2 hours at 30°C. Basal binding was measured in the absence of agonist and non-specific binding was measured in the presence of 20 μM unlabeled GTPγS. The reaction was terminated by vacuum filtration though Whatman GF/B glass fiber filters, followed by three washes with 4°C Tris buffer (50 mM Tris-HCl, pH 7.4). Bound radioactivity was determined by liquid scintillation spectrophotometry at 95% efficiency after 10-h extraction in ScintiSafe Econo 1 scintillation fluid.
