Tissue was placed in 5 mL cold membrane buffer (50 mM Tris-HCl, 3 mM MgCl2, 1 mM EGTA, pH 7.4) and homogenized. URB597 (50 nM) was then incubated with the homogenate for 30 min at 30°C, in order to ensure that there was significant inhibition of FAAH before anandamide was added to the protein homogenate. The samples were then centrifuged at 50,000 g at 5°C for 10 min. The supernatant was removed and samples were re-suspended in 5mL assay buffer A (50 mM Tris-HCl, 3 mM MgCl2, 0.2 mM EGTA, 100 mM NaCl, pH 7.4). Protein concentration was determined by Bradford method (Bradford, 1976). Prior to assay, membranes (4–8 μg protein) were pre-incubated for 25 min at 30°C with adenosine deaminase (3 μU/ml) in assay buffer A. Concentration-effect curves were generated by incubating the appropriate amount of membrane protein (4–8 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
