Western blot: Technique, theory, and trouble shooting

Mahmood, Tahrin; Yang, Ping‐Chang

doi:10.4103/1947-2714.100998

Cited by 906 publications

(266 citation statements)

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“…Western blotting was performed according to literature [21] with some modifications. The total protein of the cells was extracted using 500µl Lysis Buffer containing 2µl protease inhibitor Mix (Protein extract Kit, Vazyme Inc., USA).…”

Section: Methodsmentioning

confidence: 99%

Rsf-1 Influences the Sensitivity of Non-Small Cell Lung Cancer to Paclitaxel by Regulating NF-κB Pathway and Its Downstream Proteins

Chen

Sun

Guan

et al. 2017

Cell Physiol Biochem

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Background/Aims: The therapeutic efficacy of paclitaxel is hampered by chemotherapeutic resistance in non-small cell lung cancer (NSCLC). Rsf-1 enhanced paclitaxel resistance via nuclear factor-κB (NF-κB) in ovarian cancer cells and nasopharyngeal carcinoma. This study assessed the function of Rsf-1 in the modulation of the sensitivity of NSCLC to paclitaxel via the NF-κB pathway. Methods: The mRNA and protein levels of the related genes were quantified by RT-PCR and Western blotting. Rsf-1 silencing was achieved with CRISPR/Cas9 gene editing. Cell cycle, migration and proliferation were tested with flow cytometry, transwell test and CCK8 test. Cell apoptosis was analyzed with flow cytometry and quantification of C-capase3. The parameters of the tumors were measured in H460 cell xenograft mice. Results: Rsf-1 was highly expressed in H460 and H1299 cells. Rsf-1 knockout caused cell arrest at the G1 phase, increased cell apoptosis, and decreased migration and cell proliferation. Rsf-1 knockout increased the inhibition of cell proliferation, the reduction in cell migration and the augment in cell apoptosis in paclitaxel treated H460 and H1299 cells. Rsf-1 knockout further enhanced the paclitaxel-mediated decrease in the volume and weight of the tumors in H460 cell xenograft mice. Helenalin and Rsf-1 knockout decreased the protein levels of p-P65, BcL2, CFLAR, and XIAP; hSNF2H knockout decreased the protein level of NF-κB p-P65 without altering Rsf-1 and p65 protein levels, while Rsf-1 and hSNF2H double knockout decreased the level of NF-κB p-P65, in H1299 and H460 cells. Conclusion: These results demonstrate that Rsf-1 influences the sensitivity of NSCLC to paclitaxel via regulation of the NF-κB pathway and its downstream genes.

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Section: Methodsmentioning

confidence: 99%

Rsf-1 Influences the Sensitivity of Non-Small Cell Lung Cancer to Paclitaxel by Regulating NF-κB Pathway and Its Downstream Proteins

Chen

Sun

Guan

et al. 2017

Cell Physiol Biochem

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“…The epithelial tissue was scrapped with a glass slide. The epithelial tissue was then processed for the extraction of RNA and protein following published procedures [17,20]. …”

Section: Methodsmentioning

confidence: 99%

Cholera Toxin Suppresses Expression of Ubiquitin Editing Enzyme A20 and Enhances Transcytosis

Zhu

Wang

2013

Cell Physiol Biochem

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Background: The ubiquitin editing enzyme A20 plays an important role in maintaining the homeostasis in the body Microbe-derived adjuvants are commonly used in animal models of intestinal allergy. Objective: This study aims to investigate the role of cholera toxin-induced A20 suppression in compromising intestinal barrier function. Methods: Human intestinal epithelial cells were cultured into monolayers as an in vitro epithelial barrier model. Ovalbumin (OVA) was used as a specific allergen to test the degrading capability of intestinal epithelial cells for the endocytic allergens. The fusion of endosomes and lysosomes in epithelial cells was observed by immunocytochemistry. The antigenicity of OVA was tested by T cell proliferation assay. Results: A20 was detectable in the intestinal cell lines and mouse intestinal epithelialum. A20 was required in the degradation of endocytic allergens in HT-29 cells. The allergen, OVA, could pass through A20-deficient HT-29 monolayer barrier. Exposure to microbial adjuvant, cholera toxin, suppressed the expression of A20 in HT-29 cells, which compromised the epithelial barrier function. Conclusion: The microbial product, cholera toxin, interferes with the expression of A20 in intestinal epithelial cells, which compromises the intestinal epithelial barrier function.

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“…Western blot procedure was performed as described previously 26 using anti‐integrin‐α v (BD Biosciences, Franklin Lakes, NJ, USA) and anti‐β‐actin (Sigma‐Aldrich, St. Louis, MO, USA).…”

Section: Methodsmentioning

confidence: 99%

Deoxycholic acid promotes development of gastroesophageal reflux disease and Barrett's oesophagus by modulating integrin‐αv trafficking

Prichard

Byrne

Murphy

et al. 2017

J Cellular Molecular Medi

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The fundamental mechanisms underlying erosive oesophagitis and subsequent development of Barrett's oesophagus (BO) are poorly understood. Here, we investigated the contribution of specific components of the gastric refluxate on adhesion molecules involved in epithelial barrier maintenance. Cell line models of squamous epithelium (HET‐1A) and BO (QH) were used to examine the effects of bile acids on cell adhesion to extracellular matrix proteins (Collagen, laminin, vitronectin, fibronectin) and expression of integrin ligands (α3, α4, α5, α6 and αν). Experimental findings were validated in human explant oesophageal biopsies, a rat model of gastroesophageal reflux disease (GORD) and in patient tissue microarrays. The bile acid deoxycholic acid (DCA) specifically reduced adhesion of HET‐1A cells to vitronectin and reduced cell‐surface expression of integrin‐αν via effects on endocytic recycling processes. Increased expression of integrin‐αv was observed in ulcerated tissue in a rat model of GORD and in oesophagitis and Barrett's intestinal metaplasia patient tissue compared to normal squamous epithelium. Increased expression of integrin‐αν was observed in QH BO cells compared to HET‐1A cells. QH cells were resistant to DCA‐mediated loss of adhesion and reduction in cell‐surface expression of integrin‐αν. We demonstrated that a specific component of the gastric refluxate, DCA, affects the epithelial barrier through modulation of integrin αν expression, providing a novel mechanism for bile acid‐mediated erosion of oesophageal squamous epithelium and promotion of BO. Strategies aimed at preventing bile acid‐mediated erosion should be considered in the clinical management of patients with GORD.

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Western blot: Technique, theory, and trouble shooting

Cited by 906 publications

References 0 publications

Rsf-1 Influences the Sensitivity of Non-Small Cell Lung Cancer to Paclitaxel by Regulating NF-κB Pathway and Its Downstream Proteins

Rsf-1 Influences the Sensitivity of Non-Small Cell Lung Cancer to Paclitaxel by Regulating NF-κB Pathway and Its Downstream Proteins

Cholera Toxin Suppresses Expression of Ubiquitin Editing Enzyme A20 and Enhances Transcytosis

Deoxycholic acid promotes development of gastroesophageal reflux disease and Barrett's oesophagus by modulating integrin‐αv trafficking

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