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2 protocols using mouse tnf α

1

Protein Extraction and Western Blot Analysis

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As described previously (Wang et al., 2017 (link)), the freshly colonic tissues were excised and fragmented using a scalpel. 500 ug–1 mg tissue portions were lysed for 30 min on ice with RIPA lysis buffer (Beyotime) in the presence of cocktail protease inhibitor (Roche) and phoSTOP phosphatase inhibitor (Roche). An additional step of sonication was also performed. Protein extracts (35 µg) were boiled and subjected to 10% SDS-PAGE gel before transfer to nitrocellulose membranes. The membranes were blocked for 2 h in PBST (PBS with 0.5% Tween 20) with 5% non-fat dry milk (Bio-Rad) and incubated with specific primary antibodies at 4°C overnight. Appropriate HRP-conjugated second antibodies (Cell Signaling Technology, #7076) were used at 1:3,000 dilution for 2 h at room temperature. Signals were detected by ECL HRP substrate (Advansta). The following primary antibodies were used: mouseβ-actin (Santa Cruz, sc-47778), mouse NF-κB p65 (Cell Signaling Technology, #6956), and mouse TNF-α (Abcam, ab1793).
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2

Anti-inflammatory Molecular Mechanisms

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Hyperin (purity > 98%) was purchased from Preferred (Chengdu, China). LPS (Escherichia coli 055:B5) and Dimethyl sulfoxide (DMSO) was provided from Sigma Chemical Co. (St.Louis, MO, USA). Mouse TNF-α, IL-6 and IL-1β ELISA kits were purchased from Abcam (Cambridge, UK). Mouse NLRP3, ASC, caspase-1, TLR4 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). NF-κB p65, NF-κB p-p65, IκB, p-IκB, and β-actin were provided from Cell Signaling Technology Inc. (Beverly, MA). All other chemicals were of reagent grade.
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