SDS-PAGE and Western blotting

SDS-PAGE was performed according to the standard protocols published elsewhere [38] with minor modifications. Briefly, cells were lysed after incubation in 1% SDS with 1:1000 protease inhibitior cocktail (Sigma, Taufenkirchen, Germany). After sonication, the protein concentration was determined by using a modified Lowry method (DC^™ Protein Assay Kit, Bio-Rad, California, USA). 4x SDS-Page sample buffer (40% glycerol, 20% beta-mercaptoethanol, 12% SDS, 0.4% bromphenol blue) was added, and after heating the samples (20 μg total protein/lane) were applied to 12% (w/v) SDS-polyacrylamide gels. After electroblotting, immunodetection was carried out (1:1000 dilution of primary antibodies, 1:20,000 dilution of secondary antibody). Antigen-antibody complexes were visualized by an enhanced chemiluminescence system. Beta-actin and TOM-20 were used as internal control for equal loading.