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Binding studies were carried out for 30 min at 37°C with 20 to 40 μg of membranes with subtype-specific radioligands (12). Receptor coupling to adenylyl cyclase was tested by incubating cells for 30 min with 1 μM forskolin with or without ligand at 37°C (12).
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D-A)
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The photobleaching decay was analyzed for the plasma membrane region on a pixel-by-pixel basis as well as averaged over the entire image. Image analysis procedures and instrumental setup are described in (9). CHO-K1 cells stably cotransfected with HA-SSTR5 and D2R were grown on glass cover slips for 24 hours, treated with agonists or antagonists for 30 min at 37°C, fixed, and processed for immunocytochemistry. HA-SSTR5 and D2R were specifically labeled with FITC and rhodamine, respectively, using mouse monoclonal HA antibodies and rabbit polyclonal antibodies followed by reaction with conjugated secondary antibodies. Both reactions resulted in specific plasma membrane staining.
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We thank H. H. Niznik for providing the cDNA for the long form of D2R, K. Koller for the cDNA for HA-SSTR5, and M. Correia for secretarial help. Supported by NIH grants NS32160-05 and NS34339 and Canadian Medical Research Council (MRC) grant MT-10411. Y.C.P. is a Distinguished Scientist of the MRC. M.R. is supported by a studentship from the Fonds de la Recherche en Santé du Quebec.
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