β2AR stable cells were lysed on ice in 50 mM Tris-HCl, pH 7.7 for 10 minutes, then scraped and pelleted by centrifugation at 1,000 rpm for five minutes. The pellet was washed with 50 mM Tris-HCl, pH 7.7 and passed through a 22-gauge needle five times. Lysed cells were spun at 27,000 × g for 10 minutes and the pellet was resuspended in buffer containing 15 mM Tris-HCl, pH 7.4, 120 mM sodium chloride, 5.4 mM potassium chloride, 1.8 mM calcium chloride, and 5 mM glucose. Total membrane protein concentration was determined by Bradford assay (Bio-Rad, Hercules, CA, USA).
Thirty micrograms of β2AR stable cell membrane protein were incubated with 3 nM [3H]dihydroalprenolol ([3H]DHA, PerkinElmer) in the presence of increasing concentrations of agonist (10−10 to 10−3 M) in a final volume of 1 ml. Non-specific binding was determined with 10 μM alprenolol. The mixture was incubated at 25 °C for 2 hours and then filtered over 25 mm Whatman GF/C glass microfiber filters (GE Healthcare, Chicago, IL, USA) soaked in 0.05% polyethyleneimine. The filters were washed 3 times with ice cold Tris buffered saline. Bound radioactivity was measured with a TriCarb 4910 TR liquid scintillation analyzer (PerkinElmer) and expressed as % of maximum [3H]DHA specific binding.
Thirty micrograms of β2AR stable cell membrane protein were incubated with 3 nM [3H]dihydroalprenolol ([3H]DHA, PerkinElmer) in the presence of increasing concentrations of agonist (10−10 to 10−3 M) in a final volume of 1 ml. Non-specific binding was determined with 10 μM alprenolol. The mixture was incubated at 25 °C for 2 hours and then filtered over 25 mm Whatman GF/C glass microfiber filters (GE Healthcare, Chicago, IL, USA) soaked in 0.05% polyethyleneimine. The filters were washed 3 times with ice cold Tris buffered saline. Bound radioactivity was measured with a TriCarb 4910 TR liquid scintillation analyzer (PerkinElmer) and expressed as % of maximum [3H]DHA specific binding.