反应详情

Measurements with [3H]DPCPX in the absence of GTP were performed according to a protocol published previously.30 Adenosine A2A receptor affinities were determined according to Gao et al.31 Adenosine A3 receptor affinities were determined essentially as described.28,29 Briefly, assays were performed in 50/10/1 buffer (50 mM Tris/10 mM MgCl2/1 mM ethylenediaminetetra-acetic acid (EDTA) and 0.01% 3-([3-cholamidopropyl]-dimethylammonio)-1-propanesulfonate (CHAPS)) in glass tubes and contained 50 μl of a HEK 293 cell membrane suspension (10–30 μg), 25 μL [125I]AB MECA (final concentration 0.15 nM), and 25 μl of ligand. Incubations were carried out for 1 hr at 37° C. and were terminated by rapid filtration over Whatman GF/B filters, using a Brandell cell harvester (Brandell, Gaithersburg, Md.). Tubes were washed three times with 3 ml of buffer. Radioactivity was determined in a Beckman 5500B γ-counter. Nonspecific binding was determined in the presence of 10−5 M R-PIA.

1. customwere terminated by rapid filtration over Whatman GF/B filters
2. washTubes were washed three times with 3 ml of buffer