Claudin 1

Manufactured by Santa Cruz Biotechnology

Sourced in United States, Canada

Claudin-1 is a protein involved in the formation of tight junctions between cells. It plays a crucial role in maintaining the integrity and permeability of epithelial and endothelial cell barriers. Claudin-1 is a key component of the tight junction complex and is essential for regulating the movement of molecules across cell layers.

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Lab products found in correlation

28 protocols using claudin 1

Immunostaining of Intestinal Markers

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Prolong Gold antifade with 4′,6-diamidino-2-phenylindole (DAPI) was obtained from Life Technologies (Grand Island, NY). FITC-tagged UEA-1 lectin was obtained from Sigma, St. Louis, MO (cat. L9006; 10 μg/mL). The following antibodies were used as primary antibodies: NKCC1 (cat. ab59791, 1:200; Abcam); CLCA1 (Developmental Studies Hybridoma Bank 10.1.1 monoclonal, 1:100); mucin 2 (cat. sc-7314; 1:100; Santa Cruz Biotechnology, Dallas, TX); claudin-1 (cat. 71-7800, 1:200; ThermoFisher, Waltham, MA); claudin-2 (cat. 35-5600, 1:200; ThermoFisher); and Bio-Plex Pro Mouse Cytokine kit (cat. M6000007NY; Bio-Rad, Hercules, CA).

Koumangoye R., Omer S., Kabeer M.H, & Delpire E. (2019). Novel Human NKCC1 Mutations Cause Defects in Goblet Cell Mucus Secretion and Chronic Inflammation. Cellular and Molecular Gastroenterology and Hepatology, 9(2), 239-255.

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Protein Expression Analysis by Western Blot

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Protein expression was assessed by western blot analysis. Protein (50 μg) samples were resolved by 10% SDS–PAGE and transferred to PVDF membranes, then probed with various primary antibodies against E-cadherin, claudin-1, and vimentin (Santa Cruz Biotechnology); FN (Sigma-Aldrich, St Louis, MO, USA); SIP1 and SOX4 (Abcam Trading Company, Shanghai, China). After incubating with an appropriate secondary antibody, the protein expression was detected by western blot analysis, as described previously.^{27 (link)–29 (link)}β-Actin (Santa Cruz Biotechnology) was used as an internal control.

Xiao L., Zhou X., Liu F., Hu C., Zhu X., Luo Y., Wang M., Xu X., Yang S., Kanwar Y.S, & Sun L. (2015). MicroRNA-129-5p modulates epithelial-to-mesenchymal transition by targeting SIP1 and SOX4 during peritoneal dialysis. Laboratory investigation; a journal of technical methods and pathology, 95(7), 817-832.

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Quantifying Protein Expression in Cell Lysates

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Protein concentration was measured with a Pierce^™ BCA protein assay (Thermo Fisher Scientific, IL, USA) according to the manufacturer’s instructions. Proteins in cell lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described previously [39 (link)]. After transfer, membranes were blocked by 5% non-fat milk (Cat. M7409, Sigma Aldrich) for 1 h at 20–21°C. Blocked membranes were then incubated overnight at 4°C with primary antibodies from ProteinTech (Rosemont, IL, USA): zonula occludens-1 (ZO-1, 20742–1-AP), ZO-2 (18900–1-AP), ZO-3 (22116–1-AP), occludin (13409–1-AP), claudin-1 (13050–1-AP), claudin-4 (16195–1-AP), Cyp1a1 (13241–1-AP), heme oxygenase-1 (HO-1, 10701–1-AP), Nrf2 (16396–1-AP), beta-actin (20536–1-AP) and lamin A/C (10298–1-AP) or from Santa Cruz Biotechnology (Dallas, TX, USA): AhR (sc-133088). After a 1-h incubation with anti-rabbit IgG, horseradish peroxidase (HRP)-conjugated (#7074, CST) or anti-mouse IgG, HRP-conjugated (7076, CST) secondary antibodies, blot images were captured using a FluorChem E imager (ProteinSimple, San Jose, CA). The intensities were quantified by densitometric analysis using AlphaView software for FluorChem^™ systems.

Tocmo R., Le B., Heun A., Pijkeren J.P., Parkin K, & Johnson J.J. (2020). Prenylated xanthones from mangosteen (Garcinia mangostana) activate the AhR and Nrf2 pathways and protect intestinal barrier integrity in HT-29 cells. Free radical biology & medicine, 163, 102-115.

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Colon Tissue Protein Extraction and Analysis

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Total protein in the colon tissues was homogenized in ice-cold RIPA lysis buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% sodium deoxycholate, 0.1% SDS, 10 mM NaF, 5 mM EDTA, 1 mM Na₃VO₄, and 1% protease inhibitor (Roche) for 1 min, incubated for 30 min on ice and centrifuged at 14000 g for 10 min. Protein concentration of liquid supernatant was determined using BCA assay kit (Thermo Scientific Pierce). The proteins were denatured, loaded on polyacrylamide gel (10 μg/well), separated by electrophoresis, and then transferred onto the PVDF membrane. This membrane was blocked with 5% defatted milk in TBST for 1 h and incubated at 4 °C overnight with primary antibodies against β-actin (internal control, Cell Signaling Technology), Bax, Bcl-2, ZO-1, ZO-2, claudin-1, or occluding (Santa Cruz). Subsequently, the membranes were incubated with HRP-conjugated secondary antibodies (Cell Signaling Technology), and blots were developed using the ECL detection reagents (Millipore) and analyzed by Image J software (Rawak Software, Inc. Germany).

Yu X.T., Xu Y.F., Huang Y.F., Qu C., Xu L.Q., Su Z.R., Zeng H.F., Zheng L., Yi T.G., Li H.L., Chen J.P, & Zhang X.J. (2018). Berberrubine attenuates mucosal lesions and inflammation in dextran sodium sulfate-induced colitis in mice. PLoS ONE, 13(3), e0194069.

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Protein Interaction and Stability Analysis

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The antibodies listed below were utilized: USP40 (cat# sc-514248, Santa Cruz), USP40 (ab121234, Abcam), Claudin1 (cat# 13255, Cell Signaling Technology), Claudin1 (cat# sc-166338, Santa Cruz), Claudin1 (cat# 13050-1-AP, Proteintech), HA-tag (cat# 51064-2-AP, Proteintech), c-Myc (ab32072, Abcam), KLF4 (cat# 12173, Cell Signaling Technology), Ub (cat# sc-8017, Santa Cruz), and β-actin (cat# 66009-1-Ig, Proteintech). Cycloheximide (CHX, cat# S7418) and MG132 (cat# S2619) were purchased from Selleck.

Wu Q., Qiu Y., Guo J., Yuan Z., Yang Y., Zhu Q., Zhang Z., Guo J., Wu Y., Zhang J., Huang D., Tu K, & Hu X. (2024). USP40 promotes hepatocellular carcinoma cell proliferation, migration and stemness by deubiquitinating and stabilizing Claudin1. Biology Direct, 19, 13.

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Western Blot Analysis of Gut Markers

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Proteins were separated by SDS gels and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked with 3% bovine serum albumin prepared in TBS (Tris-buffered saline, pH 7.5 containing 0.1% Tween-20) for 1 h and then incubated with antibodies overnight at 4 °C: anti-p65 (1:200. Santa Cruz, CA, USA), anti-IκBα (1:200. Santa Cruz, CA, USA), IKKβ (1:200. Santa Cruz, CA, USA), mucin-2 (Muc-2, 1:500. Abcam, Cambridge, MA, USA), intestinal trefoil factor (ITF, 1:500. Abcam, Cambridge, MA, USA), ZO-1 (1:500. Abcam, Cambridge, MA, USA), Occludin (1:500. Abcam, Cambridge, MA, USA), HIF-1α (1:500. Santa Cruz, CA, USA), HIF-1β (1:500. Santa Cruz, CA, USA), Cleaved-caspase-3 (1:500. Santa Cruz, CA, USA), Claudin-1 (1:500. Abcam, Cambridge, MA, USA), and anti-GAPDH (1:1000. Santa Cruz, CA, USA). The membranes were then washed three times in TBST (50 mM Tris-HCl pH 7.5, 140 mM NaCl, 0.1% Tween) and incubated with secondary antibody at room temperature for 1 h. An enhanced chemiluminescence reagent, ECL Western blotting detection reagent (Tanon, Shanghai, China), was used to make the labeled protein bands detectable with Image System Minichemi (Saizhi, Beijing, China).

Wu C., Wang X., Jiang T., Li C., Zhang L., Gao X., Tian F., Li N, & Li J. (2016). Partial Enteral Nutrition Mitigated Ischemia/Reperfusion-Induced Damage of Rat Small Intestinal Barrier. Nutrients, 8(8), 502.

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Protein analysis of rat ileums

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Total protein was extracted from rat ileums, and the concentration of total protein was determined by BCA Protein Assay kit (TransGen, Beijing, China). Western blotting was performed with the following primary antibodies: ACSL4 (Abcam), GPX4 (Abcam), FTH1 (Abcam), ZO-1 (Abcam), occludin (Zymed Laboratories), claudin-1 (Santa Cruz Biotechnology), and claudin-2 (Zymed Laboratories). β-Actin (Sigma) was used as loading control. For protein quantification, the density of Western blot bands was measured by Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, MD, USA).

Ma D., Jiang P., Jiang Y., Li H, & Zhang D. (2021). Effects of Lipid Peroxidation-Mediated Ferroptosis on Severe Acute Pancreatitis-Induced Intestinal Barrier Injury and Bacterial Translocation. Oxidative Medicine and Cellular Longevity, 2021, 6644576.

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Molecular Markers in Cell Signaling

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Antibodies to p-STAT3, STAT3, p-Src, Src,p-NFκBp65,NF-κBp65, cleaved-caspase-3, cleaved-PARP were purchased from Cell Signaling Technology (Danvers, MA). Antibodies to E-cadherin, CD68, MCP-1, Claudin-1, Claudin-4, MMP-2, MMP-9 and GAPDH were purchased from Santa Cruz, CA. The Tunel assay kit for apoptosis was purchased from Roche Life Science (Indianapolis, IN). Antibodies to neutrophil gelatinase-associated lipocalin (NGAL) were purchased from R&D Systems (Minneapolis, MN). Antibodies to α-tubulin, and all other chemicals were obtained from Sigma (St. Louis, MO).

Xiong C., Zang X., Zhou X., Liu L., Masucci M.V., Tang J., Li X., Liu N., Bayliss G., Zhao T.C, & Zhuang S. (2017). Pharmacological inhibition of Src kinase protects against acute kidney injury in a murine model of renal ischemia/reperfusion. Oncotarget, 8(19), 31238-31253.

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Evaluating Intestinal Barrier Integrity

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Thinned and mature apples were obtained from an orchard located in Gyeongsan, Gyeongsangbuk-do, Korea. To estimate the integrity and paracellular permeability of intestinal junctions, transwell plates were purchased from Corning (New York, NY, USA). Fluorescein isothiocyanate-dextran (FITC-dextran, MW 4000 Da) and all other chemicals were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Cell culture media (Dulbecco’s modified Eagle’s medium, DMEM), trypsin, fetal bovine serum (FBS), phosphate-buffered saline (PBS), and related reagents were purchased from Welgene (Daegu, Korea) and Gibco (Grand Island, NY, USA). Claudin-1, claudin-4, claudin-5, ZO-1, and occludin primary antibodies were purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). Horseradish peroxidase-conjugated secondary antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA).

Lee J.Y, & Kim C.Y. (2022). Preventive Effects of Thinned Apple Extracts on TNF-α-Induced Intestinal Tight Junction Dysfunction in Caco-2 Cells through Myosin Light Chain Kinase Suppression. Foods, 11(12), 1714.

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Immunohistochemical Analysis of Tight Junctions

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The tissue sections were deparaffinized and high-pressure antigen repair was performed with citric acid repair solution. After cooling to room temperature, endogenous peroxidase in slices was inactivated with 3% H₂O₂ solution. After blocking the non-specific antigen with 5% BSA, primary antibodies were added and placed at 4°C for at least 14 h. Main primary antibodies were Occludin (1:100, Abcam, Cambridge, United Kingdom), Claudin 1 (1:200, Santa Cruz, CA, United States), Claudin 5 (1:200, Zen-Bioscience, Chengdu, China). The corresponding secondary antibody was incubated at room temperature for 1 h. After staining with DAB and hematoxylin, the slices were sealed and placed under an optical microscope (Olympus, Shanghai, China) for observation and image collection. IHC images were analyzed by Image-Pro-Plus7.0 software.

Li Y., Liu J., Pongkorpsakol P., Xiong Z., Li L., Jiang X., Zhao H., Yuan D., Zhang C., Guo Y, & Dun Y. (2022). Relief Effects of Icariin on Inflammation-Induced Decrease of Tight Junctions in Intestinal Epithelial Cells. Frontiers in Pharmacology, 13, 903762.

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