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2 protocols using rabbit polyclonal anti phospho stat3

1

Investigating JAK2/STAT3 Signaling Pathways

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Unless otherwise stated, all chemicals, including aspirin (ASA), LPS (Escherichia coli 055:B5), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), and mouse monoclonal anti-β-actin, were obtained from Sigma Chemical Co., St. Louis, MO, USA. A BCA protein assay kit and a Revert Aid First Strand cDNA Synthesis Kit were purchased from Thermo Scientific, Waltham, MA, USA, and TRIZOL Reagent from Life technologies, Carlsbad, CA, USA. Rabbit monoclonal anti-phospho-JAK2, rabbit monoclonal anti-JAK2, rabbit polyclonal anti-phospho-STAT3, mouse monoclonal anti-STAT3, rabbit monoclonal anti-phospho-P65, and rabbit monoclonal anti-P65 antibodies were supplied by Cell Signaling Technology, Inc., Danvers, MA, USA, and mouse monoclonal anti-IRP1 was from Abcam, San Francisco, CA, USA. Goat anti-rabbit and anti-mouse IRDye 800CW secondary antibodies were bought from LI-COR bio sciences, Lincoln, NE, USA. The Health Department of Hong Kong and Shanghai Government and the Animal Research Ethics Committee of The Chinese University of Hong Kong (the project identification code: GRF14106914, 1 January 2015) and Fudan University (the project identification code: NSFC31271132, 1 January 2013; NSFC31330035, 1 January 2014) approved the experimental procedures of this study.
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2

Protein Extraction and Western Blot Analysis

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For the preparation of total cell extracts, tissue samples were homogenized in RIPA buffer (50 mMTris-HCl, pH 7.4, 150 mM NaCl, 1% Triton-X100, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with 1 mM PMSF and 1% protease and phosphatase inhibitor cocktail (Sigma-Aldrich). Protein concentration was determined using the Bradford method [38] . Equivalent amounts (50 µg) of protein were subjected to electrophoresis on a 4-12% Mini-PROTEAN® TGX™ Gel (Bio-Rad) and transferred to a Hybond ECL nitrocellulose membrane (GE Healthcare Bio-Science). The following primary antibodies were used: rabbit polyclonal anti-GAPDH (1:5000, Sigma-Aldrich), rabbit polyclonal anti-STAT3 (1:1000, Sigma-Aldrich), rabbit polyclonal anti-phospho-STAT3 (1:1000, Cell Signaling Technology), and rabbit polyclonal anti-BDNF (1:500, Santa Cruz Biotechnology). An HRP-conjugated goat anti-rabbit IgG (1:5000, Jackson ImmunoResearch Laboratories) secondary antibody was used. Densitometric analysis of digitized images was performed using Chemidoc XRS Imaging Systems and Image LabTM Software (Bio-Rad).
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