Proteins were extracted from 1 × 107 cells from the three HNSCC cell lines and protein concentration was determined with the Pierce BCA protein assay kit (Fisher Scientific, Schwerte, Germany). For Western blot analyses, 50 μg protein/lane was separated on 8%-12% SDS-PAGEs, transferred onto nitrocellulose membranes (Schleicher and Schüll, Dassel, Germany) and stained with (3%, w/v) Ponceau S. Immunodetection was performed with specific primary mAbs directed against TAP1, TAP2, LMP2, LMP10, HLA-I HC and β2-m (kindly provided by S. Ferrone, Harvard, Boston, MA, USA) as recently described [8 (link)]. Staining of blots with a GAPDH mAb (Cell Signaling, Frankfurt, Germany) served as loading control. As secondary antibodies the horseradish peroxidase (HRP)-conjugated anti-rabbit and anti-mouse antibodies (DAKO, Hamburg, Germany), respectively, were used, and the LumiLight WB substrate (Roche Diagnostics, Mannheim, Germany) was employed for detection and visualization with a CCD camera (Eastman Kodak Co., Berlin, Germany).
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