Radioligand binding and competition assays

HEK-β2AR were pelleted by centrifugation at 1,000 x g for 10 minutes. Cells were re-suspended in assay buffer (7.5 mM Tris-HCl and 5 mM EDTA, pH 7.0) and homogenized 20 times. Membranes were pelleted via centrifugation at 40,000 x g for 30 minutes at 4 °C and re-suspended in assay buffer at 1 µg/µl protein with DC Protein Assay (BioRad Laboratories, Hercules CA). For competition binding assays, 50 µg of membrane was incubated with 0.8 nM of [³H]-Dihydroalprenolol (Perkin Elmer, Waltham, MA) and varying concentrations of isoproterenol in the presence or absence of 100 µM H₂O₂ and/or 1 mM dimedone. Non-specific binding was determined as the amount of radioligand bound in the presence of 10 μM propranolol. 4 mM magnesium chloride was added to the binding buffer to elucidate the effects of divalent cations. CALU3 membranes were prepared in the same manner in 25 mM HEPES with 5 mM EDTA (pH 7.0). Saturation isotherms were used to quantify the maximal binding (B_max) using approximately 100 µg of protein and a saturating concentration of [³H]-Dihydroalprenolol (10 nM) with or without 1 mM H₂O₂ and/or 1 mM dimedone, as noted in the figure legends. All reactions were incubated for 30 minutes at room temperature with agitation and terminated by rapid filtration over Whatman filters, washed with iced assay buffer supplemented with 0.1% BSA and assessed by liquid scintillation counting.