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5 protocols using claudin 2

1

Quantitative Protein Analysis in Membranes

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Protein concentration was determined by the BCA protein assay [43 (link)]. Membranes were exposed to the following antibodies purchased from Cell Signaling Technology: Occludin (#91131), Claudin-2 (#48120), Cleaved-IL-1β (#63124), IL-18 (#57058), Caspase-1 (#2225), NLRP3 (#15101), ASC/TMS1 (#67824), AIM2(#63660), β-actin (#4970), Malt1(#2494), Bcl10(#4237), Nod1(#3545), Card9 (#12283), Ripk2 (#4142). Anti-ZO-1 antibody purchased from abcam (ab221547), Anti-NLRC4 antibody purchased from ECM Bioscience(#NP5381). Western blotting signals were quantified by a FluorChem densitometer (Alpha Innotech, San Leandro, CA).
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2

Quantitative Analysis of Intestinal Proteins

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All the buffers were supplemented with protease inhibitors (AGScientific) during enterocyte separation. Solubilized proteins were separated by 10% SDS-PAGE. Visualization was by chemiluminescence (SuperSignal, Thermo Fisher, 34580) and digital imaging (Protein Simple, San Jose,). Rabbit anti-mouse ZIP14 antibody was custom-made by Genscript (Piscataway, NJ) (47 (link)). Β-Actin (Cell Signaling, 8457), Claudin 1 (ThermoFisher, 71-7800), Claudin 2 (Cell Signaling, 48120), ZO-1 (ThermoFisher, 61-7300), P-EGFR (Cell Signalling, 3777), P-AKT (Cell Signalling, 4060), AKT (Cell Signalling, 4691), P-STAT3 (Cell Signalling, 9145), STAT3 (Cell Signalling, 30835), Cleaved Caspase 3 (Cell Signalling, 9661), SLC39A4/ZIP4 (Proteintech, 20625-1), SLC39A5/ZIP5 (Novusbio, NBP3-04949). Band intensities were measured by the software ImageJ. Values for protein of interests were normalized to their control (pan-proteins or β-actin). The percentage of changes over control groups was presented.
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3

Protein Expression Analysis in Colon Tissues

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WB analysis was performed as described previously 15 (link). In brief, after quantification, proteins extracted from cells or colon tissues were separated by SDS-PAGE gels, transferred to nitrocellulose membranes, and blocked with 5% BSA. After incubation with primary antibodies and corresponding HRP-conjugated secondary antibodies, the protein bands were visualized using an ECL substrate (BIO-RAD, 1705061, Hercules, CA, USA). The primary antibodies used for the WB are listed as follows: GAPDH (Cell Signaling Technology, 2118S), P2Y1R (Affinity, DF10258), p-AMPK (Bioworld Technology, BS5003), AMPK (Bioworld Technology, BS1009), ZO-1 (Abcam, ab96587), occludin (Abcam, ab222691), and claudin-2 (Cell Signaling Technology, 48120).
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4

Epithelial-Mesenchymal Transition Markers

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Proteins were isolated from cells treated with no treatment,.5 mM ATP for hours or 6 hours, or 10 ng/ml TGF-β for 2 hours or 6 hours. Proteins were analyzed with western blots using appropriate primary rabbit anti-human antibodies all purchased from Cell signaling Technologies: E-cadherin (# 3195), Snail (#3879), Vimentin (# 5741), MMP-1 (#54376), MMP-3 (# 14351), MMP-9 (# 2270), Claudin-2 (# 48120), and NF-κBp65 (# 4764). Secondary antibody staining was completed with anti-rabbit IgG, HRP-linked antibody (Goat, 1:1000, CST, #7074). Cofilin (D3F9) XP® Rabbit mAb (#5175) was used as a protein loading control. The signals were detected with Super Signal West Pico Chemiluminescent substrate (Thermo Fisher Scientific) and was developed using (Odyssey Fc 2800, LI-COR Biosciences). Intensities of protein bands were quantified by the corresponding Odyssey Fc software used to develop blots.
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5

Epithelial-Mesenchymal Transition Protein Analysis

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Proteins were isolated from cells treated with no treatment, .5 mM ATP for 2 hours or 6 hours, or 10 ng/ml TGF-b for 2 hours or 6 hours. Proteins were analyzed with western blots using appropriate primary rabbit anti-human antibodies all purchased from Cell signaling Technologies: E-cadherin (# 3195), Snail (#3879), Vimentin (# 5741), MMP-1 (#54376), MMP-3 (# 14351), MMP-9 (# 2270), Claudin-2 (# 48120), and NF-kBp65 (# 4764). Secondary antibody staining was completed with anti-rabbit IgG, HRP-linked antibody (Goat, 1:1000, CST, #7074). Co lin (D3F9) XP® Rabbit mAb (#5175) was used as a protein loading control. The signals were detected with Super Signal West Pico Chemiluminescent substrate (Thermo Fisher Scienti c) and was developed using (Odyssey Fc 2800, LI-COR Biosciences). Intensities of protein bands were quanti ed by the corresponding Odyssey Fc software used to develop blots.
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