Whole-cell lysates of CD73-shRNA-treated cells were extracted with 300 μL of radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris, 150 mM sodium chloride (NaCl), 1% NP40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate (SDS)) and subjected to Western blotting analysis. The membranes were then incubated with polyclonal antibodies against CD73 (ab91086, 1:1000; Abcam, Cambridge, UK), E-cadherin (GTX124178, 1:5000; Genetex, Irvine, CA, USA), vimentin (ab92547, 1:1000; Abcam, Cambridge, UK), snail (#3879, 1:1000; Cell Signaling, Danvers, MA, USA), and β-actin (A5441, 1:10000; Sigma-Aldrich, St. Louis, Missouri, USA). Horseradish peroxidase-conjugated anti-rabbit secondary antibody was added to detect the primary antibodies, and the blots were developed using a chemiluminescence system (Pierce). All resolved protein bands were developed using the Western Lightning Chemiluminescence Reagent Plus system (Amersham Biosciences). All experiments were repeated at least three times, with similar results. The whole Western Blot figures can be found in the Supplementary Materials File .
E cadherin
Manufactured by GeneTex
Sourced in United States, United Kingdom
E-cadherin is a transmembrane protein that plays a crucial role in cell-cell adhesion. It is responsible for the formation and maintenance of adherens junctions, which are essential for the structural and functional integrity of epithelial tissues.
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Lab products found in correlation
Vimentin, by GeneTex (18 mentions)
N cadherin, by GeneTex (14 mentions)
N cadherin, by Abcam (5 mentions)
β actin, by Merck Group (4 mentions)
Pvdf membrane, by Merck Group (4 mentions)
β actin, by GeneTex (4 mentions)
Gapdh, by GeneTex (3 mentions)
Twist, by GeneTex (3 mentions)
Snail, by Cell Signaling Technology (3 mentions)
Twist1, by GeneTex (3 mentions)
Snail, by GeneTex (3 mentions)
β actin, by Cell Signaling Technology (3 mentions)
Western lightning chemiluminescence reagent plus system, by Cytiva (2 mentions)
Ab92547, by Abcam (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Gapdh, by Proteintech (2 mentions)
β actin, by Abcam (2 mentions)
H3k27me3, by Active Motif (2 mentions)
Quantity one software, by Bio-Rad (2 mentions)
Protease inhibitor cocktail, by Solarbio (2 mentions)
43 protocols using e cadherin
1
Western Blot Analysis of CD73 Knockdown
2
Protein Expression Analysis Protocol
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Protein extraction was performed using lysis buffer (Beyotime). Equal amounts of proteins were isolated with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). After blockage with 5% fat-free milk, membranes were incubated with primary antibodies against N-cadherin (1:1,500; GeneTex, Irvine, CA, USA), E-cadherin (1:3,000; GeneTex), Vimentin (1:5,000; GeneTex), GAPDH (1:5,000; GeneTex), FLOT2 (1:1,000; GeneTex) at 4°C overnight, and then incubated with horseradish peroxidase-conjugated secondary antibodies (1:2,000; GeneTex) for 1 h. Protein levels were measured by enhanced chemiluminescence solution (Beyotime).
3
2D-DIGE Analysis of Galectin-1 Signaling
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Fluorescent dyes (Cy2, Cy3, and Cy5) and reagents for 2D-DIGE were purchased from GE Healthcare (Uppsala, Sweden). Lipofectamine® RNAiMAX Transfection Reagent and OPTI-MEM were purchased from Invitrogen (Waltham, MA, USA). MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was purchased from USB Corp. (Cleveland, OH, USA). Propidium iodide (PI) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Recombinant human LGALS1 protein (rhLGALS1) was purchased from BioLegend, Inc. (San Diego, CA, USA). LGALS1, cyclin A2, cdk2, phosphor-ERK (Thr202), MMP-9, MMP-3, ZEB2, SNAI1, TWIST, E-cadherin, N-cadherin, and vimentin primary antibodies were purchased from Genetex Inc. (Hsinchu, Taiwan). Phospho-p38 MAPK (Thr180/Tyr182) and p38 MAPK primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Cyclin D2, cdk4, cyclin E, and p27 primary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). ERK1/2 primary antibody was purchased from Promega Corp. (Madison, WI, USA). Anti-rabbit and anti-mouse immunoglobulin (Ig)G secondary antibodies were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA, USA). All the chemicals and reagents used in this study were of analytic grade.
4
Protein Extraction and Immunoblotting
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Total cellular protein was extracted by cells lysis buffer RIPA, which was added to a protease inhibitor mixture. Next, 30 ug of protein was separated by SDS-PAGE, and the protein was transferred onto a polyvinylidene fluoride (PVDF) membrane by the wet transfer method, and the protein – antibody complex was detected by immunoassay(19). Primary antibodies used were as follows: TET1 (GeneTex, 1:1000), E-cadherin (GeneTex, 1:1000), Vimentin (GeneTex, 1:1000), N-cadherin (GeneTex, 1:1000), Beclin1 (GeneTex, 1:1000), LC3 (Beyotime, AF5225, 1:500), P62 (Beyotime, AF5312,1:500), and Atg5 (Beyotime, 1:500); GAPDH (Beyotime, AF0006, 1:1000). The second antibodies included HRP-goat anti-rabbit (Beyotime, AF0208, 1:1000) or HRP-goat anti-mouse (Beyotime, AF0216, 1:1000). Finally, the protein – antibody complexes were detected with an enhanced chemiluminescence (ECL) reagent.
5
Western Blot Analysis of EMT Markers
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For Western blot analysis, the cells were harvested and lysed in 1X RIPA buffer containing protease inhibitors and phosphatase inhibitors (Roche, Mannheim, Germany). Protein concentration was determined using a Bio-Rad DC protein assay kit (Bio-Rad, Hercules, CA, USA). Total protein (20 μg) was loaded onto a 10% SDS-polyacrylamide gel for electrophoresis. Specific antibodies against E-cadherin (GTX100443, GeneTex, Irvine, CA, USA), N-cadherin (ab12221, Abcam, Cambridge, USA), Vimentin (ab92547, Abcam), Snail (#3879, Cell Signaling Technology, Inc., Danvers, MA), Slug (GTX30813, GeneTex), and Twist1 (GTX100621, GeneTex) were applied to detect the targets. Monoclonal anti-Actin antibody (Sigma, St. Louis, MO, USA) was used as the loading control. The following secondary antibodies were used: goat anti-rabbit or anti-mouse immunoglobulin G (IgG)-conjugated horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA, USA). To detect the specific signals, the membranes were examined using the ECL Prime Western Blotting Detection System (GE Healthcare, Waukesha, WI, USA).
6
Quantitative Analysis of Epithelial-Mesenchymal Markers
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Transfected cells were lysed using RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific) with a mixture of protease inhibitors. The total protein concentration was determined using the Bradford method. We separated 20 μg of total protein using SDS-PAGE, and then transferred it onto PVDF membranes (Millipore, USA). Immunoblots were blocked with 5% non-fat dried milk for 1 h at room temperature. After blocking, membranes were incubated with antibodies for IQGAP1 (1: 1000; Abcam, USA), E-cadherin (1: 1000; GeneTex, USA), N-cadherin (1: 1000; Abcam), and β-actin (1: 2000, Santa Cruz) separately overnight at 4°C. Secondary antibodies (1: 2000, Cell Signaling Technology, USA) were then used for incubation for 2 h at room temperature. Results were visualized using an ECL kit (Tanon, Shanghai, China).
7
Immunohistochemical Analysis of E-Cadherin and Vimentin
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For IHC analyses, excised tumors were fixed in formalin and embedded in paraffin. All tumor samples were 4 μm thick tissue sections. These sections were treated with heat-induced antigen retrieval at 95 °C in sodium citrate buffer (10 mM, pH 6.0) for 30 min. Endogenous peroxidase was blocked by 3% hydrogen peroxide (H2O2) at room temperature for 30 min. Nonspecific reaction were blocked by BlockPRO™ Blocking Buffer (Visual Protein, Taipei, Taiwan) at room temperature for 30 min. Sections were then incubated with primary antibody against E-Cadherin (1: 250; GeneTex, Irvine, CA USA) and vimentin (1: 50; Cell Signaling, Denver, MA, USA) overnight at 4 °C, followed by using VECTASTAIN® ABC Kit (Rabbit IgG)( PK-6101; Vector Laboratories, Peterborough, UK). Sections were stained with Liquid DAB+ Substrate Chromogen System (DAKO, Carpinteria, CA, USA) and counterstained with hematoxylin. An oncology pathologist expert helped in examining the stained slides and estimating the expression levels of E-Cadherin and vimentin. The IHC staining was then observed and photographed under light microscope.
8
Protein Expression Analysis of Tumor Samples
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Tumors were resected from the tumor-bearing mice after 4 weeks of 188 Re-liposome treatment, and lysed using the T-PER™ Tissue Protein Extraction Reagent (Thermo Fisher Scienti ic, Waltham, MA) containing 1% of protease inhibitor cocktail (Sigma-Aldrich). Protein lysates were run on 8% -12% sodium dodecyl sulfate -polyacrylamide gel electrophoresis (SDS-PAGE). The fractionated proteins https://doi.org/10.29328/journal.hor. 1001024 were transferred based on previously [19] . The antibodies included E-cadherin (GTX100443), Slug (GTX128796), ZEB-1(GTX105278), vimentin (GTX100619), Snail (GTX100754) were from GeneTex (Genetex Inc. Irvine, CA, USA). Antibody against GAPDH (MA5-15738) was from Sigma (Sigma-Aldrich Co, St. Louis, MO, USA).
9
Comprehensive Histone and EMT Marker Analysis
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Primary antibodies for H3K4me3 (GTX128954), H3K9me3 (GTX121677), H3K27me3 (GTX121184), H3K36me3 (GTX54109), H3K79me3 (GTX54111), histone H3 (GTX122148), MMP2 (GTX104577), MMP3 (GTX100723), MMP9 (GTX100458), N-cadherin (GTX101141), E-cadherin (GTX100433), vimentin (GTX100619), Snail (GTX100754), TWIST-1 (GTX60776), Nanog (GTX100863), OCT4 (GTX101497), SOX2 (GTX101507), ABCB1 (GTX108354), ABCG2 (GTX100437) and ABCC1 (GTX88673) were purchased from GeneTex International Corporation (Hsinchu City, Taiwan). Anti-mouse and anti-rabbit IgG-conjugated horseradish peroxidase and rabbit polyclonal antibodies speci c for β-actin (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany; cat. no. SI-A5441; 1:10,000) were used in this study. All other chemicals were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany).
10
Nasal Mucosal Protein Expression Analysis
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Equal amounts of nasal mucosal proteins (40 μg) extracted by the aforementioned method were subjected to electrically separated in 10% polyacrylamide gel, and then transferred to polyvinylidene difluoride membrane. Because of limited amount of nasal tissue retrieved from the experimental mice, we cut the polyvinylidene difluoride membrane, before blocking and incubating with individual primary antibody. The membrane was incubated with the following primary antibodies: claudin-1 (1:1000, catalog No. E-AB-30939, Elabscience, Houston, USA), e-cadherin (1:2000, catalog No. GTX100443, GeneTex, Irvine, CA, USA), MAC5AC (1:100, clone No. 2-11M1, abcam, Cambridge, UK), and GAPDH (1:5000, catalog No. 60004-1-1 g, Proteintech, Rosemont, USA). On the following day, the membranes were incubated with HRP-conjugated secondary antibody, followed by electrochemiluminescent detection (Millipore Billerica, MA, USA). The density of each protein band was scanned using ImageJ Software, version 1.46r (National Institutes of Health, Bethesda, MD) and compared in densitometry.
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