Membrane Preparation
Membrane fractions were prepared from transfected HEK293 cells as described previously (Zöllner, 2003). The cells were grown in 175 cm2 tissue culture flasks to approximately 90 % confluence. Cells were then washed with Tris buffer (50 mM, Trizma preset crystals, pH 7.4; Sigma Aldrich, Darmstadt, Germany), harvested with a scraper, homogenized using a mechanical disperser (Dispergierstation T8.10, IKA-Werke, Staufen, Germany) at maximum speed for 10 s and centrifuged at 42K x g for 20 min at 4oC (Avanti JXN-26 ultracentrifuge, Beckmann Coulter, Krefeld, Germany). Cellular pellets including membranes with embedded and anchored proteins were then resuspendend in Tris buffer for washing to separate them from cytosolic components by homogenization and centrifugation at the same settings. Supernatants were discarded and the pellets were stored at -80 oC. On the day of usage, the pellets were thawed on ice in Tris buffer and homogenized. Total protein concentrations were determined according to the Bradford method (Bio-Rad Laboratories GmbH, München, Germany). (Bradford, 1976) and homogenates were split according to the number of conditions tested in respective assay buffers.