Proteins were extracted using RIPA lysis buffer containing 1% PMSF and cocktail (Beyotime, Haimen, China). The protein concentrations were measured by BCA assay. The proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking with non-fat milk (5%), the membranes were then incubated overnight at 4 °C with the following antibodies: anti-DNA-PKcs (abcam, ab32566, 1:5000), anti-vimentin (CST, 5741, 1:1000) anti-E-cadherin (bioworld, bs1098, 1: 1000), anti-intergrin-β4 (abcam, ab182120, 1:1000) and anti-GAPDH (CST, 5174, 1:1000). The membranes were washed thrice with TBST buffer and then incubated with secondary antibodies at room temperature for 1 h. Enhanced chemiluminescence (ECL) reagent (Thermo Fisher Scientific, Inc.) was used for the detection of interest protein bands. Image J v1.48u software (National Institutes of Health, Bethesda, MD) was employed to analyze the relative optical densities of interest bands.
Anti e cadherin
Manufactured by Bioworld Technology
Sourced in United States, United Kingdom
Anti-E-cadherin is a laboratory reagent used in various cell and molecular biology applications. It is a monoclonal antibody that specifically binds to the E-cadherin protein, which is a key component of cell-cell adhesion complexes. The primary function of Anti-E-cadherin is to detect and quantify the expression of E-cadherin in biological samples.
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Lab products found in correlation
Anti vimentin, by Bioworld Technology (6 mentions)
Anti n cadherin, by Bioworld Technology (4 mentions)
Anti mmp 2, by Bioworld Technology (3 mentions)
Anti mmp 9, by Bioworld Technology (3 mentions)
Goat tritc labeled secondary antibody, by Bioworld Technology (2 mentions)
Nitrocellulose membrane, by Merck Group (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ab32566, by Abcam (1 mentions)
Ab182120, by Abcam (1 mentions)
Ecl reagent, by Thermo Fisher Scientific (1 mentions)
Ab129450, by Abcam (1 mentions)
Ab3373, by Abcam (1 mentions)
Ab24102, by Abcam (1 mentions)
Nc membrane, by Merck Group (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Irdye goat igg, by LI COR (1 mentions)
Trans blot system, by Bio-Rad (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Gapdh, by Proteintech (1 mentions)
16 protocols using anti e cadherin
1
Protein Expression Analysis Protocol
2
Immunofluorescence and Western Blot Analysis
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For immunofluorescence assay, cells were grown on glass slices and fixed in 4 % formaldehyde for 10 min, permeabilized through 0.3 % Triton X-100. Then the slices were blocked in goat serum for 15 min, 37°C and incubated overnight at 4°C with anti-E-cadherin (1:80, Bioworld, MN, USA), anti-N-cadherin (1:80, Bioworld, MN, USA). Samples were washed three times before incubated with goat TRITC labeled secondary antibody (1:70, Bioworld, MN, USA) at 37°C for 1 h. DAPI (Genview Inc, Shanghai, China) was used for counterstaining. Fluorescence was visualized with a microscope under ×400 magnification.
Total protein was extracted using RIPA Lysis Buffer (Beyotime, China) and PMSF (Sigma-Aldrich). The proteins were transferred to NC membranes (Millipore Corp, MA USA) using the TransBlot System (Bio-Rad, CA, USA). The membranes were blocked in 5% w/v non-fat milk in TBS and incubations were performed overnight at 4°C. The membranes were then washed using TBST and incubated with secondary antibodies (1:10000, IRDye Goat IgG, LI-COR Bioscience, NE USA) for 1h at room temperature. Protein staining was detected using the Odyssey Imaging System (LI-COR Biosciences, NE USA). The following primary antibodies were used: GAPDH (1:10000, Proteintech Group, Chicago USA), ABCC1 (1:100, Abcam, ab24102), ABCC2 (1:100, Abcam, ab3373), P-gp1 (1:2000, Abcam, ab129450).
Total protein was extracted using RIPA Lysis Buffer (Beyotime, China) and PMSF (Sigma-Aldrich). The proteins were transferred to NC membranes (Millipore Corp, MA USA) using the TransBlot System (Bio-Rad, CA, USA). The membranes were blocked in 5% w/v non-fat milk in TBS and incubations were performed overnight at 4°C. The membranes were then washed using TBST and incubated with secondary antibodies (1:10000, IRDye Goat IgG, LI-COR Bioscience, NE USA) for 1h at room temperature. Protein staining was detected using the Odyssey Imaging System (LI-COR Biosciences, NE USA). The following primary antibodies were used: GAPDH (1:10000, Proteintech Group, Chicago USA), ABCC1 (1:100, Abcam, ab24102), ABCC2 (1:100, Abcam, ab3373), P-gp1 (1:2000, Abcam, ab129450).
3
Protein Expression Analysis in HK-2 Cells
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Proteins (25 μg) isolated from HK-2 cells were separated using 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The membranes were treated with 10% fat-free milk in Tris-buffered saline prior to incubating with primary antibodies, including anti-E-cadherin (1:800; Bio-world, Visalia, CA, USA), anti-α-SMA (1:2000; ProteinTech, Rosemont, IL, USA), anti-tight junction protein 1 (TJP1; 1:1000; CST, Danvers, MA, USA), anti-fibronectin (1:500; WanleiBio), anti-Notch1 antibody (1:500; WanleiBio), and anti-Jagged-1 antibody 1:500; WanleiBio). The membranes were then incubated with horseradish-peroxidase-conjugated secondary antibody (1:5000; Santa, Dallas, TX, USA). Protein signals were then visualized using an Enhanced chemiluminescence (ECL) kit, then normalized to β-actin.
4
Western Blot Analysis of Epithelial-Mesenchymal Transition
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Protein was extracted from kidney tissue and cultured cells. The protein concentration was determined using the Bradford method. Then, 40 μg of the extracted protein was electrophoresed on 10 % SDS polyacrylamide gels and transferred to nitrocellulose membranes (Millipore, Bedford, MA, USA). After blocking for 2 h at 4 °C in blocking buffer (10 % fat-free milk TBS with 0.1 %Tween 20), the membrane was incubated overnight with anti-E-cadherin (1:800, Bio-world), anti-α-SMA (1 μg/ml, Abcam), and anti-β-tubulin (1: 5000, Bio-world). The membrane was washed and incubated for 2 h at 4 °C with conjugated anti-rabbit secondary antibody. Detection was performed using enhanced chemiluminescence (Thermo Scientific, Frederick, MD, USA) and photography.
5
Protein Extraction and Western Blot Analysis
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Proteins were extracted using 1% SDS lysis buffer. The concentration of proteins was measured with the Pierce BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Each sample was separated by SDS-PAGE gel, transferred into nitrocellulose membrane (Millipore Corporation, Billerica, MA, USA). Membranes were incubated with the following primary antibodies: anti-PPP3CB (Abcam, Cambridge, UK), anti-E-cadherin (Bioworld, Nanjing, China), anti-Vimentin (Cell Signaling Technology, Danvers, MA, USA), anti-N-cadherin (Abcam, Cambridge, UK), anti-Twist1(Abcam, Cambridge, UK),anti-Snail1(Cell Signaling Technology, Danvers, MA, USA),anti-GFP (Bioworld, Nanjing, China), anti-β-actin (Transgene, Beijing, China). The target bands were detected by ECL (Millipore Corporation, Billerica, MA, USA).
6
Immunofluorescence Staining of Cadherins
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Cells were cultured on glass slides and fixed with 4% formaldehyde for about 10 min. Next, 0.3% Triton X-100 was used for cell permeation. The slices were blocked by the goat serum for about 15 min at 37 °C. Subsequently, samples were incubated with anti-E-cadherin (1:80, Bioworld, MN, USA) and anti-N-cadherin (1:80, Bioworld, MN, USA) at 4 °C overnight and with goat TRITC-labeled secondary antibody (1:70, Bioworld, MN, USA) at 37 °C for 1 h. Meanwhile, DAPI (Genview Inc., Shanghai, China) was utilized for staining. At last, the fluorescence was visualized under a microscope (×400).
7
Western Blot Analysis of Cell Signaling
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The si-NC and si-CHD5 siRNA were transfected into U87 cells with Lipofectamine 2000 reagent. After the cells were transfected for 48 h, Western blot analysis was performed as previously described [53 (link)]. Briefly, the cells were lysed with 1% SDS lysing buffer containing protease inhibitor cocktail and phosphatase inhibitor cocktail (Apexbio, Houston, TX, USA). The protein concentration was determined using the BCA protein assay reagent kit (Thermo Scientific, Waltham, MA, USA). All the blots were incubated with the respective primary antibodies, namely, anti-β-Tubulin (1:5000, TRANSGEN, Beijing, China), anti-CDK4, anti-CDK6, anti-CDK9 (1:800, Cell Signaling Technology, Boston, MA, USA), anti-E-cadherin, anti-N-cadherin, and anti-Twist1 (1:1000, Bioworld, Nanjing, China) antibodies. The protein bands were visualized with ECL Reagents (Smart-Lifesciences, Nanjing, China).
8
TNBS-Induced Intestinal Fibrosis Treatment
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Curcumin and 2,4,5-trinitrobenzene sulphonic acid (TNBS) were purchased from Sigma (USA); rosiglitazone was kindly provided by Huanghe Medical Share Co. Ltd. (China). Anti-E-cadherin was from Bioworld (USA) and ELISA kit of FN from Bluegene (USA); Anti-Smad2/3, anti-pSmad3, anti-PPARγ were purchased from Cell Signaling Technology (USA); PPARγ antagonist GW9662 was from Selleckchem (USA); IEC-6 cell line was obtained from American Type Culture Collection, ATCC. Dulbecco's modified Eagle's medium (DMEM) and foetal bovine serum (FBS) were purchased from Biological Industries (Israel) and all other chemicals were from AMRESCO (USA). Sprague-Dawley (SD) rats were purchased from Matter Technology (China).
9
Immunohistochemistry Staining of DACH1, E-Cadherin, Vimentin, MMP-2, and MMP-9
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Immunohistochemistry staining (IHC) was performed in 32 cases of available matched cancer and adjacent non-cancerous tissue samples. The procedure was performed as described previously [19 (link)]. Anti-DACH1 with 1/500 dilution (Proteintech, Chicago, IL, USA), anti-E-cadherin with 1/50 dilution (Bioworld Technology, Beijing, China) and anti-vimentin, anti-MMP-2, anti-MMP-9 (Bioworld Technology) with 1/100 dilution were incubated overnight at 4°C. The staining intensity and extent of the staining area were graded according to the German semi-quantitative scoring system as described before [19 (link)]. Staining intensity of the nucleus, cytoplasm and/or membrane (no staining = 0; weak staining = 1; moderate staining = 2; strong staining = 3); extent of stained cells (0% = 0, 1–24% = 1, 25–49% = 2, 50–74% = 3, 75–100% = 4). The final immunoreactive score (0–12) was determined by multiplying intensity score to the extent of stained cells score.
10
Western Blot Analysis of TGF-β Pathway Proteins
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Cell lysate was prepared using RIPA buffer with protease inhibitors and quantified using the BCA protein assay (BioTek, China). Protein (20 μg) was loaded onto a 10% SDS-PAGE gel that was then transferred onto PVDF membrane and incubated with anti-TGFβR2 (Bioworld Technology, MN.US), anti-TGF-β1 (Cell Signaling Technology), anti-PI3K (Cell Signaling Technology), anti-p-Akt (p-Ser473, Abzoom), anti-c-myc (Santa Cruz), anti-E2F1 (Santa Cruz), anti-CCND1 (Santa Cruz), anti-p21 (Santa Cruz), anti-E-cadherin (Bioworld Technology, MN.US), anti-Vimentin (Bioworld Technology, MN.US), and anti-Snail (Bioworld Technology, MN.US) at 4°C overnight in blocker (3% non-fat dry milk/BSA in TTBS) followed by incubation with HRP-conjugated secondary anti mouse (ZSGB-Bio, China). Protein was normalized with GAPDH (Abmart).
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