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2 protocols using egfr rabbit mab

1

Immunohistochemical Analysis of mTOR, S6, and EGFR

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For immunohistochemistry analysis, 4-μm sections were depa-raffinized through graded ethanol series, rehydrated, and washed with Tris-buffered saline (TBS) containing 0.1% (w/v) Tween 20. Sections were then incubated overnight at 4 °C with the following primary antibodies at the indicated dilution: (1) phospho-mTOR (Ser2448) (49F9) rabbit mAb (1:50) (Cell Signaling Technology, Beverly, MA, USA), (2) phospho-S6 ribosomal protein (Ser235/ 236) (91B2) rabbit mAb (1:75) (Cell Signaling Technology), (3) EGFR rabbit mAb (1:400) (Cell Signaling Technology). Antibody binding was detected by the avidin-biotin complex (ABC) method of staining using a Vectastain Elite ABC kit (PK-6101; Vector Laboratories, Burlingame, CA, USA) for which the Dako EnVison Plus HRP system (K4006) was used following the manufacturer's protocol.
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2

Recombinant Protein Expression in E. coli

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The E. coli strain JM-109 (Promega) was used for plasmid proliferation and cloning. The E. coli strain Rosetta-gami 2 (Novagen) was used as a host for the production of recombinant proteins. Human nasopharyngeal carcinoma cell line CNE2 and human embryonic kidney cell line (HEK 293T) were purchased from the American Type Culture Collection (ATCC). Plasmid pET-44a was purchased from Promega. All primers used in this study were synthesized by Biosune Biology and Technology Company (Shanghai, China). NdeI and XhoI restriction endonucleases, T4 DNA ligase, and PrimeSTAR HS DNA polymerase were purchased from Takara Biotechnology (Dalian, China). Human IL-2, IFN-γ, and TNF-α enzyme linked immunosorbent assay (ELISA) kits were purchased from R&D Systems (Minneapolis, Minnesota, USA). β-tubulin rabbit mAb and EGFR rabbit mAb were purchased from Cell Signaling Technology (Danvers, Massachusetts, USA). Strep-Tag II mAb was purchased from Merck (Billerica, Massachusetts, USA). (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt) (MTS) reagent for cell proliferation assay was purchased from Promega (Madison, Wisconsin, USA). Annexin V-conjugated Alexa Fluor 488 (Alexa488) Apoptosis Detection Kit was purchased from BD Pharmingen (San Diego, California, USA).
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