Gapdh mab

Proteins were extracted from cells using ice-cold RIPA lysis buffer containing 1 μM phenylmethanesulfonyl fluoride (PMSF; Sigma). Equal amounts of protein were separated by SDS-PAGE and blotted onto PVDF membranes. After blocking, the membranes were probed with an anti-Bim mAb (#2933S; 1:1000 dilution; Cell Signaling Technology, Beverly, USA) overnight at 4°C. A GAPDH mAb (#5174S; 1:1000 dilution; Cell Signaling Technology) was used as the control. Then the PVDF membranes incubated with HRP-conjugated secondary antibody (1:10000 dilution; Cell Signaling Technology) for 1 h at room temperature. The protein bands were visualized using the Enhanced chemiluminescent (ECL) detection reagents (Monad).

Cells or tissues were lysed in RIPA buffer. The following primary antibodies were used to detect the proteins of interest: (1) cyclin antibodies (Cyclin Antibody Sampler Kit, Cell Signaling, Danvers, MA, USA); (2) mTOR mAb, 4E-BP1 mAb, p70 S6 kinase mAb, and GAPDH mAb (Cell Signaling, Danvers, MA,USA); (3) mouse GFAP mAb and rabbit DCX polyclonal antibody (Santa Cruz, Dallas, TX, USA). Protein bands were visualized and optical density was analyzed using a Biospectrum 500 imaging system (UVP, LLC, Upland, CA, USA).

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).