Anti egfr monoclonal antibody

EGF was purchased from American R&D Company (Minneapolis, MN, United States). Cycloheximide (CHX) was purchased from Sigma-Aldrich Corporation (United States). Anti-human DENND1A monoclonal antibody, anti-human Grb2 monoclonal antibody, anti-human HA monoclonal antibody and anti-EGFR monoclonal antibody were purchased from Cell Signaling Technology (Danvers, MA, United States). Normal mice IgG, normal rabbit IgG and anti-GAPDH polyclonal antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States). Anti-GST antibody was purchased from cwbiotech (Beijing, China) and anti-Rab35 polyclonal antibody was purchased from Biogot Technology (Nanjing, Jiangsu, China). Anti-Flag monoclonal antibody and FITC-labeled secondary antibody were purchased from Sigma-Aldrich Corporation. TRITC-labeled and horseradish peroxidase (HRP)-labeled secondary antibodies were purchased from Jackson ImmunoResearch (United States).

Immunohistochemical staining was performed on formalin-fixed, paraffin-embedded tissue TMA sections. Briefly, 3 μm-thick TMA tissue sections were placed onto adhesive slides and treated with the PT link (DAKO) solution. Immunohistochemical staining was performed using the following primary antibodies: anti-Fatty Acid Synthase polyclonal antibody (1:100, Enzo Life Sciences), anti-EGFR monoclonal antibody (1:100, clone D38B1, CellSignaling), anti-Cytokeratin 5/6 monoclonal antibody (1:50/100, Clone D5/16 B4, DAKO) anti Monoclonal Mouse Anti-vimentin (1:100/200, Clone Vim 3B4, DAKO). Sections were washed with PBS and sequentially incubated at room temperature for 45 minutes with antirabbit or antimouse IgG. Immunodetection was performed with the kit EnVision™ (DAKO, Glostrup, Denmark) using the AutostainerPlus Link (DAKO).

OSCC, HSC-3, SCC-25, A431 and MCF-7 cells were lysed in mammalian lysate buffer (M-PER, Pierce, IL, USA) with protease inhibitor (Complete Mini, Roche, Germany). Fifty micrograms of total protein from each cell lysate samples were then resolved on a 10% bis-tris gel before transferring to a nitrocellulose membrane. The membrane was then probed with 1:200 anti-EGFR monoclonal antibody (Cell Signaling Technology, USA) and 1:400 horseradish peroxidase-conjugated antibody (Cell Signaling Technology, USA) using the iBind Western System (Life Technologies, Carlsbad, USA). The membrane was then incubated with chemiluminescent substrate (Thermo Fisher Scientific, USA) followed by detection on traditional ECL film (Amersham Hyperfilm ECL, GE Healthcare, USA). The intensity of the bands was quantified using NIH Image J (1.41o, W. Rasband, National Institute of Health, USA) software.