Membrane preparation
hGLP-2R and pcDNA3.1(+) membranes were prepared through several centrifugation steps. The cells were scraped with PBS supplemented with a cOmplete EDTA free protease inhibitor (Roche, Basel, Switzerland) and then homogenized using a Douncer homogenizer. The homogenate was centrifuged for 3 min at 500 rpm (54 g; 4°C) and subsequently the supernatant was centrifuged for 45 min at 14 500 rpm at (24 446g; 4°C). The resulting pellet was resuspended in storage buffer (20 mM HEPES buffer (pH 7.2), 0.4 mM CaCl2, 2 mM MgCl2 and cOmplete EDTA free protease inhibitor) and stored at -80°C. Protein determination was performed according to a standard Pierce BCA protein assay protocol (Thermo Scientific, Rockford, IL).