Anti β tubulin

Western blot analyses were performed as previously described [53 (link)]. Antibodies used were anti-RUVBL1 (sc-15259, Santa Cruz, 1:500), anti-RUVBL2 (gift from Matthias Gstaiger, ETH Zurich, 1:1000), anti-βTubulin (mouse monoclonal sc-5274, Santa Cruz, 1:1000), anti-TFIIH (rabbit polyclonal sc-293, Santa Cruz, 1:4000), anti-FLAG (F-3165, Sigma, 1:20000) and anti-GFP (mouse monoclonal, sc-9996, Santa Cruz, 1:500).

Cells were lysed in RIPA buffer and centrifuged at 12000×g for 5 min at 4°C. The supernatant was collected and the protein concentration was quantified by BCA assay (Applygen Technologies Inc., Beijing, China). Proteins (20 μg) were separated by SDS-PAGE, and then transferred onto nitrocellulose membranes. The membranes were incubated with anti-E-Tmod41 antibody (prepared by AbMax Biotechnology Co., Ltd, Beijing, China), anti-glycophorin A antibody (Beijing Biosynthesis Biotechnology Co. Ltd, Beijing, China), anti-GAPDH antibody, or anti-β-tubulin (Santa cruz Biotech., USA), followed by HRP-conjugated goat anti-rabbit or mouse IgG. The signals were detected by using an Enhanced Chemiluminescence Detection (ECL) kit (Evergreen, Beijing, China).

The differential abundance of some differentially expressed proteins of interest was validated by immunoblot analyses. A first validation was performed on 3 pools of proteins, using all 15 paired samples analysed by 2D-DIGE. A second validation was performed on individual proteins extracted from cancer tissues of an additional cohort of 20 patients. Ten μg of proteins were fractionated on 12% Criterion TGX Stain-Free gels and, after gel image acquisition with the Chemidoc system (BIO-RAD) and electrotransfer onto nitrocellulose membranes. Membranes were incubated with the monoclonal antibodies anti-fibrinogen β chain [1F9] (1:500; GeneTex) and anti-β-actin (1:1000, Abcam), and the polyclonal anti-β-tubulin (1:3000, Santa-Cruz). Antibody-bound proteins were detected by enhanced chemiluminescence using the Chemidoc system after incubation with ECL HRP-conjugated secondary antibodies (1:25000 dilution, GE Heathcare) and reaction with ECL Prime Western Blotting detection reagent (GE Healthcare). The image of the gel acquired before its transfer was used as control for equal protein loading among samples.

Nuclear and cytoplasmic extracts were prepared using a Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Institute of Biotechnology, Jiangsu, China) as described previously.^{11 (link)} Total proteins were extracted using RIPA lysis buffer. Fifty micrograms of cell lysates were electrophoresed with 10% SDS-PAGE. The specific primary antibodies used against each protein in the immunoblotting were as follows: anti-Drosha (1:1000; Abcam), anti-PCNA (1:800; Abcam), anti-LAMC2 (1:1000; Millipore), anti-CD82 (1:1000; CST); the antibodies anti-EGFR, anti-p-EGFR, anti-ERK1/2, anti-p-ERK1/2, anti-MMP7 and anti-GAPDH (1:1000; all from Beyotime); anti-β-tubulin (1:500; Santa Cruz Biotechnology, Santa Cruz, TX, USA) and anti-β-actin (1:1000; Boster, Wuhan, China). The appropriate horseradish peroxidase-conjugated secondary antibodies were subsequently applied. The proteins were visualized by the enhanced chemiluminescence system (Amersham, Pharmacia Biotech, Freiburg, Germany).

Anti-EphB6 was obtained from Santa Cruz (Dallas, TX, USA). Anti-phospho-Akt (Ser473), anti-phospho-S6K, anti-phospho-ERK1/2, and anti-GAPDH were purchased from Cell Signaling Technology (New England Biolabs Ltd., Whitby, ON, Canada). Anti-PARP was purchased from EMD Millipore (Billerica, Mass, USA). Anti-β-tubulin was obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Bovine serum albumin (BSA) was purchased from BioShop Canada Inc. (Burlington, ON, Canada). Doxorubicin was purchased from Tocris Bioscience (Bristol, UK). Puromycin and polybrene were bought from Sigma-Aldrich (Oakville, ON, Canada). Resazurin was purchased from R&D Systems (Minneapolis, MN, USA). Perifosine was from Invivogen (San Diego, CA, USA). Tween-20 was from Fisher Scientific (Ottawa, ON, Canada).

Following anesthesia, the brains were removed from the skull and these were dissected into the right or left hemispheres. For protein extraction, they were placed in 10 volumes of cold homogenization buffer (120 mM NaCl, 50 mM Tris, pH 7.4) with protease inhibitors (Complete Mini, Gibco, Grand Island, NY, USA) being freshly added. Tissue then was homogenized by sonicator. Concentrations of protein were checked by the Bradford method (BioRad, Richmond, CA, USA). By adding the sampling buffer, equal amount of protein, 20 μg, was loaded and separated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used with 10% polyacrylamide and 0.05% bis-acrylamide [11 (link)]. By separating proteins on the gels, they were transferred into nitrocellulose membrane. They were probed with anti-NT-3 (1 : 300, Santa Cruz, CA, USA) and anti-trkC (1 : 300, Santa Cruz, CA, USA) as primary antibody. For secondary antibody, Peroxidase anti-rabbit IgG (Vector, PI-1000, 1 : 3000 dilution) was used. As an internal control, anti-β tubulin (1 : 300, Santa Cruz, CA, USA) was checked on the sample membrane. We detected signals with enhanced chemiluminescence (Supersignal, Pierce, Rockford, IN, USA), using autoradiogram by exposing 10 to 30 min [6 (link)–8 (link)].