Extraction and Purification of Membrane Proteins

Right atrial samples were pulverized in liquid nitrogen congelation and sonicated (Brandson Sonifier 250, ICN Hubber S.A.) in 500 μL of ice-cold 10 mM Tris HCl, pH 7.4 buffer containing a protease inhibitor cocktail (Roche Molecular Systems, Belmont, CA, USA). The sonicated tissue was further homogenized using a Polytron (VDI 12, VWR, Barcelona, Spain) for three periods of 10 s each. The homogenate was centrifuged at 12,000 × g at 4 °C for 30 min. The membranes were dispersed in 50 mM Tris HCl (pH 7.4) and 10 mM MgCl₂, washed, and resuspended in the same medium as described previously [34 (link)]. Protein concentration was determined using the BCA protein assay kit (Thermo Fisher Scientific Inc., Rockford, IL, USA) and 10 μg of protein was used for immunoblotting.