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3 protocols using pure nitrocellulose membranes

1

Antioxidant and Anti-Inflammatory Pathway Evaluation

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Protease inhibitor mixture solution and ethylenediaminetetraacetic acid (EDTA) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Phenylmethylsulfonyl fluoride (PMSF) was purchased from Sigma Aldrich Co., Ltd. (St. Louis, MO, USA). 2′,7’-Dichlorofluorescein diacetate (DCFH-DA) was obtained from Molecular Probes (Eugene, OR, USA). ECL Western Blotting Detection Reagents and pure nitrocellulose membranes were supplied by GE Healthcare (Piscataway, NJ, USA). Rabbit polyclonal antibodies against Nrf2, HO-1, p-p38, p-ERK1/2, NF-κBp65, and mouse monoclonal antibodies against, and p-IκBα, COX-2, iNOS, TNF-α, IL-6, histone, and β-actin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Rabbit anti-goat, goat anti-rabbit, and goat anti-mouse immunoglobulin G (IgG) horseradish peroxidase (HRP)-conjugated secondary antibodies were acquired from Santa Cruz Biotechnology, Inc. All other chemicals and reagents were purchased from Sigma Aldrich Co., Ltd. (St. Louis, MO, USA).
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2

ASFV Protein Analysis by Western Blotting

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For western blotting analysis, p30 and CD2v proteins were analyzed by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto pure nitrocellulose membranes (GE, Boston, MA, USA), which was blocked with 1% BSA in TBST at room temperature for 2 h, and then incubated with ASFV-positive or -negative serum as primary antibody at 4 °C overnight. After incubating with HRP-conjugated anti-pig secondary antibodies for 1 h at room temperature, signals were visualised by ECL reagent (Advansta, San Jose, CA, USA).
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3

Colorectal Carcinoma Cell Line RKO Assay

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Human colorectal carcinoma cell line, RKO, was obtained from American Type Culture Collection (Manassas, Virginia, USA). Minimum essential medium (MEM) was purchased from Gibco Laboratories (Paisley, UK). Fetal bovine serum (FBS) and [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) were purchased from Sigma-Aldrich Chemical Company (Dorset, UK). Six-well flat-bottom tissue culture plates were purchased from Nunc Inc. (Hereford, UK). Phenylmethylsulfonyl fluoride (PMSF) was purchased from Sigma-Aldrich Co., Ltd. (St. Louis, MO, USA) and protease inhibitor mixture solution was obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Enhanced chemiluminescence (ECL) western blotting detection reagents and pure nitrocellulose MEMbranes were purchased from GE Healthcare (Buckinghamshire, UK). Primary antibodies for the detection of caspase 3 and β-actin and goat antirabbit and goat anti-mouse IgG horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). All chemicals and reagents were of analytical grade and purchased from Sigma-Aldrich Co., Ltd. unless otherwise specified.
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