Western blot was performed as previously described (Lam et al., 2020 (link)). Specific primary antibodies [mouse monoclonal anti-human β-actin (Sigma-Aldrich); anti-PCNA, anti-PARP (Santa Cruz Biotechnology, Inc., Santa Cruz, California, United States of America); anti-PFKP, anti-LDHB, anti-Bcl-2, anti-survivin, anti-XIAP, anti-AKT, anti-CDK2, anti-CDK4, anti-CDK7, anti-Cyclin D2, H, anti-CDK4, anti-N-cadherin, anti-β-catenin (Cell Signaling Technology, Danvers, Massachusetts, United States of America); and corresponding horseradish peroxidase (HRP)-conjugated secondary (Cell Signaling Technology)] were purchased. An enhanced chemiluminescence (ECL) kit (GE Healthcare) was used to detect protein expression. Beta-actin was selected as a reference protein (Lam et al., 2020 (link)).
Anti n cadherin
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom, China, Germany
Anti-N-cadherin is a primary antibody that recognizes N-cadherin, a calcium-dependent cell-cell adhesion glycoprotein. It is used for the detection and analysis of N-cadherin in various applications such as Western blotting, immunohistochemistry, and flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Anti e cadherin, by Cell Signaling Technology (217 mentions)
Anti vimentin, by Cell Signaling Technology (164 mentions)
Anti snail, by Cell Signaling Technology (71 mentions)
Anti slug, by Cell Signaling Technology (44 mentions)
Anti β catenin, by Cell Signaling Technology (40 mentions)
Anti gapdh, by Cell Signaling Technology (36 mentions)
Anti akt, by Cell Signaling Technology (33 mentions)
Anti β actin, by Cell Signaling Technology (29 mentions)
Pvdf membrane, by Merck Group (24 mentions)
Ripa lysis buffer, by Beyotime (16 mentions)
Anti p akt, by Cell Signaling Technology (16 mentions)
Anti zeb1, by Cell Signaling Technology (16 mentions)
Anti mmp9, by Cell Signaling Technology (16 mentions)
Anti vimentin, by Abcam (16 mentions)
Anti cyclin d1, by Cell Signaling Technology (15 mentions)
Anti β actin, by Merck Group (14 mentions)
Anti mmp2, by Cell Signaling Technology (14 mentions)
Anti stat3, by Cell Signaling Technology (14 mentions)
Anti e cadherin, by Abcam (12 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (12 mentions)
308 protocols using anti n cadherin
1
Comprehensive Protein Expression Analysis
2
Western Blot Analysis of EMT Markers
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Cells were harvested and lysed in RIPA buffer supplemented with protease inhibitors (50 mM Tris-HCl pH8, 50 mM NaCl, 0.5% NP-40). The protein concentrations were determined by the Bio-Rad (Bradford) protein assay (Bio-Rad Laboratories Inc., Hercules, CA, USA), and a total of 50 μg of protein was separated by denaturing 12% SDS-PAGE and transferred to a PVDF membrane (EMD Millipore, Billerica, MA, USA). After blocking with 5% nonfat milk in a 0.1% TBST solution for 1 hour at room temperature, membranes were then incubated with rabbit polyclonal anti-Numb (1:1,000; Abcam, Cambridge, UK), anti-E-cadherin (1:1,000; Cell Signaling Technology Inc., Danvers, MA, USA), anti-N-cadherin (1:1,000; Cell Signaling Technology), anti-Snail 1 (1:1,000; Abcam), anti-β-catenin (1:1,000; Abcam), and anti-β-actin (1:10,000; Abcam) overnight at 4°C. After washing, blots were incubated with HRP-conjugated goat anti-rabbit secondary antibodies (1:2,000; Abcam) for 1 hour. The immunoreactive proteins were visualized using ECL detection system (Pierce Biotechnology, Rockford, IL, USA). Protein levels were determined by normalization against β-actin. All experiments were conducted in triplicate.
3
Western Blot Protein Analysis Protocol
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Western blot analysis was performed as described previously.29 Briefly, aliquots of total protein (10 µg) were electrophoresed on sodium dodecyl sulfate polyacrylamide, 10% Tris‐HCl gels (Bio‐Rad Laboratories). The separated proteins were transferred to polyvinylidene difluoride membranes (Bio‐Rad Laboratories) and incubated with primary antibodies overnight at 4°C. Proteins were detected using the following antibodies: anti‐VE‐cadherin (Abcam), anti‐ZO‐1, anti‐E‐cadherin, anti‐Zinc Finger E‐Box Binding Homeobox 1 (ZEB1), anti‐N‐cadherin, anti‐Snail (Cell Signaling Technology, Danvers, MA, USA), anti‐CD13, anti‐CD133, anti‐EpCAM (Abcam) and anti‐β actin (Sigma, Tokyo, Japan).
4
Investigating GHET1-Mediated Protein Expression
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Cells were transfected with GHET1 or control vector for 48 h. After that, cells were lysed with the RIPA lysis buffer (Beyotime, Shanghai, China) and the protein concentration was determined with the BCA assay (Beyotime, Shanghai, China). The 20 μg of protein was separated by the SDS/PAGE and transferred onto the polyvinylidene fluoride membrane (Millipore, Billerica, MA, U.S.A.). After blocking with 5% non-fat milk, the membrane was incubated with the primary antibody overnight at 4°C. And then the membrane was incubated with the horseradish peroxidase labeled secondary antibody for 1 h at room temperature. The protein bands were visualized with the ECL detection kit (Beyotime, Shanghai, China). The antibodies including anti-HIF1α (#36169), anti-VHL (#68547), anti-vimentin (#5741), anti-N-cadherin (#13116) and anti-E-cadherin (#14472) were commercially purchased from Cell Signaling Technology (Danvers, MA, U.S.A.).
5
Western Blot Analysis of EMT Markers
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Cells were washed in ice‐cold PBS and sedimented at 1000 g for 10 min at RT. Pellets were suspended in RIPA buffer (10 mm Tris/HCl, pH 7.4, 1% sodium deoxycholate, 1% Triton X‐100, 0.1% SDS) containing 1 mm PMSF and protease inhibitor cocktail (Sigma‐Aldrich). Protein concentration was quantified using Bicinchoninic acid (Thermo Scientific, Rockford, IL, USA). Total proteins were separated by SDS/PAGE and transferred to Porablot NCP membranes (Macherey‐Nagel, Düren, Germany). Blots were probed with anti‐FLAG (1 : 2000; Sigma‐Aldrich), anti‐E‐cadherin (1 : 2000; Cell Signaling Technology, Danvers, MA, USA), anti‐N‐cadherin (1 : 2000; Cell Signaling Technology), anti‐Snail (1 : 2000; Cell Signaling Technology), and β‐actin (1 : 2000; Santa Cruz Biotechnology, Dallas, TX, USA) antibodies. Primary antibody binding was detected with anti‐goat IgG‐HRP (1 : 2000; Santa Cruz Biotechnology), anti‐mouse IgG‐HRP (1 : 2000; Santa Cruz Biotechnology), or anti‐rabbit IgG‐HRP (1 : 2000; Santa Cruz Biotechnology). Membranes were revealed using the EZ‐ECL chemiluminescence kit (Biological Industries, Haemek, Israel) and the ChemiDoc Touch Gel Imaging System (Bio‐Rad, Hércules, CA, USA).
6
BBSKE Regulates EMT Signaling
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1, 2- [bis (1, 2-Benzisoselenazolone-3 (2H) -ketone)] ethane (BBSKE) was obtained from the Organoselenium Research Center at Peking University, China. Human recombinant TGF-β1 was purchased from Sino Biological Inc., Beijing, China. Antibodies used for Western blotting were anti-TXN, anti-TXNRD1 (Epitomics, USA); anti-p-Akt (Ser478), anti-Akt, anti-E-cadherin, anti-N-cadherin, anti-p-GSK-3β (Ser9), anti-GSK-3β, anti-Snail, anti-Slug and anti-Twist (Cell Signaling Technology, Danvers, MA, USA), and anti-β-actin (Santa Cruz, USA). LY294002 was purchased from Beyotime Biotechnology Corporation (Haimen, China). NADPH and DTNB [5, 5′- dithiobis (2-nitrobenzoic acid)] were purchased from Sigma (St. Louis, MO, USA).
7
Protein Expression and Interaction Analysis
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Protein extracts were resolved through SDS-PAGE, detected using anti-FABP7, anti-beta-catenin, anti-CK1 alpha and anti-GSK3 beta (Abcam, Cambridge, MA, USA), anti-HA, anti-E-cadherin, anti-N-cadherin, and anti-vimentin (Cell Signaling, Danvers, MA, USA) antibodies, respectively. Blotted membranes were stripped and re-blotted with an anti-p84 rabbit monoclonal antibody (Sigma, St. Louis, MO, USA) or anti-GAPDH mouse monoclonal antibody (Abcam, Cambridge, MA, USA) as a loading control.
8
Western Blot Analysis of Protein Markers
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For the westernblot assay, cells were harvested in ice-cold PBS 48 h after transfection and lysed on ice in cold-modified radioimmunoprecipitation buffer supplemented with protease inhibitors. Protein concentration was determined using the BCA Protein Assay Kit (Bio-Rad, CA, USA) and equal amounts of protein were analyzed by SDS-PAGE. Gels were electroblotted onto nitrocellulose membranes (Millipore, WI, USA). After blocked with 5% non-fat dry milk in Tris-buffered saline containing 0.1% Tween-20 2 h, membranes were incubated at 4°C over night with primary antibody. Primary antibodies used were anti-STAT3, anti-p-STAT3, anti-MMP2, anti-BCL2, anti-E-cadherin, anti-KIRT1, anti-SNAI2, anti-N-cadherin (Cell Signaling, USA) and GAPDH (Zhong-Shan JinQiao, China). Then, membranes were incubated with respective second antibodies and detected by peroxidase-conjugated secondary antibodies using the enhanced chemiluminescence system (ECL) (Millipore, WI, USA). The experiment was repeated three times.
9
Western Blot Analysis of Protein Targets
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Western blot assay was carried out as previously described.24 Cells were washed with PBS and then lysed with RIPA lysis buffer (Solarbio, China) and protease inhibitors (Roche Applied Science, Indianapolis, Switzerland). The protein concentration was measured using the bicinchonininc acid (BCA) protein assay kit (Beyotime Biotechnology, Shanghai, China). Equal amounts of protein were subjected to 10% SDS‐polyacrylamide gel electrophoresis and transferred onto PVDF membranes. The membranes were subsequently blocked with 5% non‐fat milk for 2 hours and incubated with primary antibodies overnight at 4°C. The primary antibodies used were anti‐MGMT (1:500, Abcam, Cambridge, UK) and anti‐Survivin (1:1000, Abcam), anti‐β‐catenin, anti‐CD44, anti‐C‐Jun, anti‐C‐Myc, anti‐cyclinD1, anti‐LEF1, anti‐TCF1/TCF7, anti‐MMP7, anti‐Axin2, anti‐Met, anti‐PARP, anti‐caspase‐3, anti‐BAX, anti‐Bcl2, anti‐cleaved‐caspase‐3, anti‐E‐cadherin, anti‐N‐cadherin and anti‐Vimentin (these primary antibodies are all: 1:1000, Cell Signaling Technology, Boston, MA, USA), as well as anti‐GAPDH, and secondary antibodies were HRP‐conjugated goat anti‐mouse or goat anti‐rabbit IgG antibody (1:1000, Beyotime). All experiments were performed in triplicate.
10
Western Blot Analysis of EMT Markers
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Proteins were extracted with RIPA lysis buffer (CWBiotech, Beijing, China) on ice for 30 minutes. Protein quantity was determined using bicinchoninic acid kit purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China). Equivalent amounts of proteins (30 µg) were separated by 10% SDS-PAGE and transferred to a polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA). Membranes were blocked with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween 20 for 1 hour at room temperature and then incubated with the following specified antibodies for 1 hour at room temperature: anti-MTBP (1:2,000 dilution; Sigma-Aldrich), anti-ZEB2 (1:5,000 dilution; Abcam), anti-E-cadherin (1:5,000 dilution; Cell Signaling Technology, Danvers, MA, USA), anti-N-cadherin (1:5,000 dilution; Cell Signaling Technology), anti-β-catenin (1:5,000 dilution; Cell Signaling Technology), anti-Vimentin (1:5,000 dilution; Cell Signaling Technology), and anti-GAPDH (1:5,000 dilution; Cell Signaling Technology). The membranes were then incubated with HRP-conjugated goat anti-mouse or anti-rabbit IgG (ZSGB-BIO) for 1 hour at room temperature. Signals were visualized using an enhanced chemiluminescence reagent and captured using AI600 version 1.2.0 on Amersham Imager 600 (GE Healthcare, Chicago, IL, USA).
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