35s gtpγs

SNr tissue was rapidly dissected on ice and immediately homogenized in ice-cold 50 mM Tris-HCl (pH 7.4), 3 mM MgCl₂, and 1 mM EGTA. The homogenate was centrifuged at 45,000 × g for 10 min at 4°C, and then the pellet was homogenized in 50mM Tris-HCl, with 3 mM MgCl₂, 1 mM EGTA and 100 mM NaCl. The resulting membrane homogenate was pre-incubated with 3 mU/ml adenosine deaminase (Roche Applied Science, IN) for 10 min at 30°C, to inactivate endogenous adenosine. Then, 10 µ;g of protein per reaction was incubated in reaction buffer containing 0.1% BSA, 30 µM GDP, 0.1 nM [³⁵S]GTPγS (Perkin Elmer, OH) and 0.6 mU/ml adenosine deaminase, with either 140 µM CP55,940 or vehicle, for 45 min at 30°C. The incubation was terminated by vacuum filtration through Whatman filters (GE Healthcare), followed by three washes with 3 ml ice-cold Tris-HCl, pH 7.4. Bound [³⁵S]GTPγS was quantified using a liquid scintillation counter in vials containing isolated [³⁵S]GTPγS-bound filter paper along with 4ml of Ecoscint scintillation fluid (National Diagnostics).