Western Blot Analysis of Antigen Presentation Proteins

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).