Cell culture methods, transient expression of α1B- and β2-ARs, and membrane preparation

COS-1 and HEK293 cells [American Type Culture Collection (ATCC)] were cultured in Dulbecco’s modified Eagle’s medium containing glutamine and 5 and 10% fetal bovine serum, respectively. CHO-1 cells (ATCC) were cultured in F-12 medium containing 10% fetal bovine serum. Cells were transiently transfected with purified plasmid DNA encoding wild-type or mutant α1B- and β2-ARs, using FuGENE HD in a 1:3 ratio of DNA and FuGENE, following the manufacturer’s protocol. Cell membranes were prepared 48 hours after transfection as described previously for α1B-ARs (41) and prepared the same way for the β2-ARs but in β2 assay buffer [50 mM tris-HCl and 3 mM MgCl2 (pH 7.4)].

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