SDS-PAGE and Western blotting
Eukaryotic cell extracts were prepared by harvesting the cells in PBS (phosphate buffered saline), and subsequent lysis of the cell pellets in SDS-PAGE sample buffer (60 mM TRIS-HCl pH = 6.8, 12% glycerol, 4% SDS, 5% β-mercapto-ethanol). Protein concentrations in cell extracts were determined using BCA Protein Assay Reagent (Pierce) according to the manufacturer's instructions. 30 μg of proteins of each cell extract were loaded on 5% SDS-polyacrylamide gels. After size-separation, proteins were electrophoretically transferred to PROTEAN Nitrocellulose Membranes (Schleicher and Schuell). ECL Western blotting was performed using Western Lightning Chemiluminescence Reagent Plus (Perkin Elmer Life Sciences) according to the supplier's instructions.