Tissue samples and cells were collected and washed three times with cold PBS and then homogenized in RIPA lysis buffer (1% NP-40, 0.5% deoxycholic acid, 50 mM Tris-HCl [pH 8.0], 0.1% sodium dodecyl sulfate (SDS), 150 mM NaCl) containing a cocktail of protease inhibitors (Sigma-Aldrich, St Louis, MO, USA). Following sonication and centrifugation (13,000× g, 15 min, 4°C), the supernatant was collected. The BCA protein assay kit (Pierce, Waltham, MA, USA) was used to determine the protein concentration according to our protocol. Samples were denatured in loading buffer, separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. Anti-GATA-3 (Abcam, Cambridge, UK; diluted 1:200), anti-ZEB1 (Abcam; diluted 1:200), EMT kit (CST, Danvers, MA, USA; diluted 1:1000) and β-actin (Sigma-Aldrich; diluted 1:5000) were incubated at 4°C overnight followed by incubation with HRP-conjugated secondary antibody (Abcam; diluted 1:5000). Specific blots were visualized using the ECL kit (Thermo Fisher Scientific).
Anti zeb1
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-ZEB1 is a primary antibody that specifically recognizes the ZEB1 protein. ZEB1 is a transcription factor involved in the regulation of gene expression. The antibody can be used to detect and study the ZEB1 protein in various applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti e cadherin, by Abcam (15 mentions)
Anti vimentin, by Abcam (12 mentions)
Anti n cadherin, by Abcam (12 mentions)
Anti snail, by Abcam (8 mentions)
Anti slug, by Abcam (6 mentions)
Pvdf membrane, by Merck Group (6 mentions)
Anti gapdh, by Abcam (5 mentions)
Anti twist, by Abcam (4 mentions)
Anti β actin, by Abcam (4 mentions)
Anti zeb2, by Abcam (4 mentions)
Anti bcl 2, by Abcam (4 mentions)
Anti mmp2, by Abcam (3 mentions)
Anti β actin, by Cell Signaling Technology (3 mentions)
Anti twist1, by Abcam (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
β actin, by Merck Group (2 mentions)
Anti p stat3, by Cell Signaling Technology (2 mentions)
Anti p src, by Cell Signaling Technology (2 mentions)
Anti t stat3, by Cell Signaling Technology (2 mentions)
Anti e cad, by Abcam (2 mentions)
39 protocols using anti zeb1
1
Western Blot Analysis of EMT Markers
2
EMT Regulation and Src/STAT3 Signaling
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The study protocol was performed according to the principles of the Declaration of Helsinki and the ethics committee of the Affiliated Hospital of Jiangnan University, Wuxi, Jiangsu Province, China. Informed consent was obtained from all patients.
The following primary antibodies were purchased from Abcam (Cambridge, England): anti-E-cad, anti-Vim, anti-Snail, anti-N-cad, anti-Twist, anti-PCNA, anti-Bax, anti-Bcl-2, anti-ZEB1, anti-NEDD9, anti-Myb, and anti-GAPDH. The following primary antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA): anti-t-SRC, anti-p-SRC, anti-t-STAT3, and anti-p-STAT3. PP1, an SRC inhibitor, was purchased from Selleck Chemicals (Houston, TX, USA).
The following primary antibodies were purchased from Abcam (Cambridge, England): anti-E-cad, anti-Vim, anti-Snail, anti-N-cad, anti-Twist, anti-PCNA, anti-Bax, anti-Bcl-2, anti-ZEB1, anti-NEDD9, anti-Myb, and anti-GAPDH. The following primary antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA): anti-t-SRC, anti-p-SRC, anti-t-STAT3, and anti-p-STAT3. PP1, an SRC inhibitor, was purchased from Selleck Chemicals (Houston, TX, USA).
3
Western Blot Analysis of Epithelial-Mesenchymal Transition
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Cells were lysed with pre-cooled RIPA lysis buffer supplemented with a cocktail of proteases inhibitors (both from Sigma-Aldrich; Merck KGaA). Total protein concentration was detected using a bicinchoninic acid protein assay kit (Nanjing KeyGen Biotech Co., Ltd.). Total proteins (10 µg) were mixed with 5X SDS-PAGE loading buffer and heated at 100°C for 5 min, then, proteins were separated by 4–12% SDS-PAGE. Proteins were transferred onto 0.45-µm-thick PVDF membranes (EMD Millipore) at 4°C for 2 h. After blocking with 5% non-fat milk for 1 h at room temperature, the PVDF membranes were incubated with primary antibodies at 4°C overnight and horseradish peroxidase (HRP)-conjugated secondary antibodies at 37°C for 1 h. Protein bands were visualized using Immobilon Western Chemilum HRP substrate (EMD Millipore). The primary antibodies used were anti-GAPDH (1:10,000; KangChen Bio-tech Co., Ltd.), anti-Cyclin D1 (1:500; Abcam), anti-ZEB1 (1:500; Abcam), anti-Vimentin (1:1,000; Abcam) and anti-E-cadherin (1:2,000; Abcam). The secondary antibody used was HRP-conjugated Goat Anti-Rabbit IgG (1:20,000; SouthernBiotech).
4
Protein Expression and Western Blot Analysis
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Protein preparation and western blot assay were performed as described previously.39 Antibodies used were as follows: anti–KIF3A, anti–vimentin (Santa Cruz Biotechnology, dilution at 1:1000), anti–Cyclin D1, anti–Cyclin E1, anti–P21, anti–E2F1, anti–E‐cadherin, anti–MMP‐2, anti–ZEB1 (abcam, dilution at 1:1000), anti–MMP‐9 (Cloud‐Clone Corp, dilution at 1:1000), anti–β‐actin (TransGen Biotech, dilution at 1:4000) and anti–phospho‐Rb antibodies (Abcam, dilution at 1:5000).
5
ZEB1 ChIP-PCR Assay for GBM
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Chip assay was performed using Simple Chip Enzymatic Chromatin IP kit (Cell Signaling Technology, USA) according to the manufacturer’s instructions. Briefly, the chromatin of the GBM cells were treated with 3 μg anti-ZEB1 (Abcam). Immunoprecipitated DNA was subjected to RT-PCR using specific primers.
6
Analyzing ZEB1, E-cadherin Expression in miR-200c Transfected Cells
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SKCs were transfected with oar-miR-200c mimics (or inhibitor) and collected 48 h later. After treating with protein lysate (RIPA：PMSF=100：1), the supernatant was collected by centrifugation at 12,000 g for 5 min at 4℃. Total protein content was determined by a BCA protein assay kit (Beyotime, China). Approximately 30 μg of extracted protein with loading buffer was loaded into wells of a 12% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) for electrophoresis and then transferred to a nitrocellulose (NC) membrane (Millipore, USA). After blocking, the membranes were treated with anti-ZEB1 (1：3,000, Abcam), anti-β-actin (1：5,000, Abcam), and anti-E-cadherin (1：2,500, BD Biosciences) antibodies and incubated overnight at 4℃. Then, the membranes were treated with horseradish peroxidase-conjugated secondary antibody (Bioworld, China) for 1 h at 37℃. The protein bands were visualized using a gel imager (Bio-Rad, USA).
7
Protein Expression Analysis by Western Blotting
8
Western Blot Analysis of ZEB1 and GADPH
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RIPA buffer (Sigma-Aldrich) was used to lyze cells. The mixture was centrifuged with 12,000 g to remove insoluble components, followed by protein concentration measurement using BCA assay. Thirty micrograms of protein were separated by SDS–PAGE and transferred to PVDF membrane (Millipore, Bedford, MA, USA). PVDF membrane was blocked with non-fat milk and incubated with primary antibodies, including anti-ZEB1 (1:1,000, Abcam, Cambridge, MA), anti-GADPH (1:1,000, Abcam). After washing with TBST, HRP-conjugated goat anti-rabbit IgG (Abcam) was used to incubate the membrane at room temperature for 2 hrs. Finally, protein bands were visualized using ECL detection kit (Beyotime Biotechnology, Shanghai, China).
9
Western Blot Analysis of Epithelial-Mesenchymal Transition Markers
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Proteins were isolated from cells by Radio Immunoprecipitation Assay (RIPA Beyotime, Nantong, China) buffer, electrophoresed on 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (Millipore, Billerica, MA, USA). After 1 hour blockage in 5% skim milk, membranes were incubated with anti-GAPDH (Beyotime, Nantong, China), anti-SHPRH, anti-E-cadherin, anti-N-cadherin, anti-ZEB1 and anti-β-actin (Abcam, Cambridge, MA, USA) at 4oC overnight. At the other day, blots were incubated with secondary anti-body (Beyotime, Nantong, China) for 1 h. Band visualization was conducted using the enhanced chemiluminescence reagent kit (Millipore, Billerica, MA, USA).
10
Western Blot Analysis of Cell Signaling
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The whole-cell extract was obtained in RIPA buffer in the presence of standard protease and phosphatase inhibitors. The protein content was determined with a protein assay reagent (Bio-Rad Laboratories Inc., Hercules, CA, USA), using bovine serum albumin as a standard. Equal protein content of total cell lysates was resolved on polyacrylamide gel (Bolt 4–12% Bis-Tris Plus Invitrogen, Carlsbad, CA, USA), electro-transferred to PVDF membranes (iBlot Invitrogen, Carlsbad, CA, USA) and incubated with specific primary antibodies: anti-YAP (Santa Cruz Biotechnology, Inc., Dallas, TX, USA; sc #101199; dil.1:1000); anti-p-YAP (Cell Signaling Technology, Inc., Beverly, MA, USA; #13008; dil.1:1000); anti-β Actin (MP Biomedicals, Santa Ana, CA, USA; # 69100; dil.1:10000); anti-Rac1, anti-RhoA and anti-Cdc42 (all Cell Biolabs, San Diego, CA, USA; STA-405; dil.1:500); anti-Zeb1 (Abcam, Cambridge, MA, USA; #180905; dil.1:2000); anti-Snail1 (Santa Cruz Biotechnology, Inc., Dallas, TX, USA; sc-271977; dil.1:1000); anti-GSN (Sigma-Aldrich, St Louis, MO, USA; G-4896, dil.1:1000). Membranes were developed using ECL detection reagents (GE Healthcare Life Sciences, Uppsala, Sweden) on a UVITEC imaging system (UVITEC, Cambridge, UK) or ChemiDocMP system (Bio-Rad Laboratories Inc.). Quantitative analysis of western blots was performed using ImageJ (NIH) software [23 (link)].
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