Total protein was isolated from differently treated cells using RIPA lysis buffer (Beyotime Institute of Biotechnology), and the total protein concentration was measured using a BCA protein assay kit. Subsequently, the protein samples (20 µg/lane) were separated using 10% SDS-PAGE, transferred to PVDF membranes and blocked with 5% skimmed milk. After blocking at 37°C for 1 h, the membranes were incubated with anti-β-catenin (catalogue number, 17565-1-AP; 1:1,000), anti-c-Myc (catalogue number, 10828-1-AP; 1:1,000), anti-cyclin D1 (catalogue number, 26939-1-AP; 1:1,000) and anti-GAPDH (catalogue number, 10494-1-AP; 1:2,000) antibodies, all from ProteinTech Group, Inc., or the aforementioned anti-HSP70, anti-TSG101, anti-CD9 and anti-calnexin antibodies at 4°C overnight. After washing, the membranes were incubated with goat anti-rabbit/mouse IgG (H+L)-HRP secondary antibody (catalogue number, 111-035-003/115-035-003; 1:5,000; Jackson ImmunoResearch Laboratories, Inc.) at 37°C for 2 h. After washing with PBST (1,000 ml 1X PBS + 1 ml Tween-20) five times, the protein bands were visualized using a Millipore ECL system (Tanon Science & Technology Co., Ltd.). The protein bands were analyzed using Image-Pro Plus software (version 6.0; Media Cybernetics Imaging Technologies Inc.).
Anti β catenin
Manufactured by Proteintech
Sourced in United States, China
Anti-β-catenin is a laboratory reagent used in various research applications. It is a monoclonal antibody that specifically binds to the β-catenin protein, which is a key component of the Wnt signaling pathway. The antibody can be used to detect and quantify the expression levels of β-catenin in biological samples.
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Lab products found in correlation
Anti gapdh, by Proteintech (13 mentions)
Anti β actin, by Proteintech (11 mentions)
Anti n cadherin, by Proteintech (9 mentions)
Anti e cadherin, by Proteintech (8 mentions)
Anti c myc, by Proteintech (7 mentions)
Anti vimentin, by Proteintech (7 mentions)
Anti cyclin d1, by Proteintech (6 mentions)
Pvdf membrane, by Merck Group (6 mentions)
Ripa lysis buffer, by Beyotime (3 mentions)
Anti mmp2, by Proteintech (3 mentions)
Anti hif 1α, by Abcam (3 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (3 mentions)
Anti gapdh antibody, by Proteintech (3 mentions)
Protease inhibitor cocktail, by Roche (3 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Anti c myc, by Cell Signaling Technology (2 mentions)
Anti p stat3, by Cell Signaling Technology (2 mentions)
Anti mmp9, by Proteintech (2 mentions)
Anti p akt, by Proteintech (2 mentions)
Protease inhibitor cocktail, by Selleck Chemicals (2 mentions)
53 protocols using anti β catenin
1
Protein Extraction and Western Blot Analysis
2
Western Blotting Analysis of Cell Signaling Pathways
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Western blotting analysis was performed using the same procedure demonstrated previously7 (link). Briefly, the radioimmunoprecipitation assay lysis buffer (Beyotime, China) was used to extract total cell protein, which was then quantified using the bicinchoninic acid protein assay kit (Beyotime, China) and resolved on a 6–12% sodium dodecyl sulphate-polyacrylamide gel, and then incubated with relevant primary antibodies. GAPDH and histone (ZSGBBIO, China) were used as loading controls. The following primary antibodies were utilised in this research: anti-PPARδ (Proteintech), anti-CXCR4 (Proteintech, China), anti-vimentin (Proteintech), anti-GAPDH (Proteintech), anti-β-catenin (Proteintech), anti-Slug (Proteintech) and anti-N-cadherin (Proteintech). We used GSK3787 (Abcam) and XAV-939 (Abcam, UK) as PPARδ and β-catenin inhibitors, respectively. The enhanced chemiluminescence system (Bio-Rad, Hercules, EDA USA) was used to detect protein expression levels and Scion imaging software was used to capture images.
3
Comprehensive Protein Expression Analysis
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Western blot analysis was conducted as previously described [24 (link)]. The following primary immunoblotting antibodies were used: anti-GAPDH, anti-E-cadherin, anti-Fibronectin, anti-Vimentin, anti-Snail, and anti-c-Met (Epitomics, Burlingame, CA, USA), anti-β-catenin (Proteintech, Chicago, USA), anti-N-cadherin, anti-c-myc, anti-EGFR, anti-p-EGFR (Tyr1068), anti-GSK3β, anti-p-GSK3β (Ser9), anti-AKT (pan), anti-p-AKT (Ser473), anti-stat3, anti-p-stat3 (Tyr705) and anti-CREB1 (Cell Signaling Technology, Beverly, MA).
4
Immunohistochemical Analysis of Protein Expression
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Paraffin-embedded samples were dewaxed with xylene and rehydrated with graded ethanol. The samples were incubated overnight at 4°C with the following primary antibodies (all 1:100; ProteinTech Group, Inc.): Anti-β-catenin (cat. no. 51067-2-AP), c-Myc (cat. no. 10828-1-AP), cyclin D1 (cat. no. 60186-1-lg), Wnt3a (cat. no. HZ-1296) and vimentin (cat. no. 10366-1-AP). Following primary incubation, samples were incubated with universal secondary antibodies (HRP-labeled). Finally, the positive detection rate was used to score the IHC results and staining intensity. Further details of the IHC analysis are presented in Data S1.
5
Western Blot Analysis of BCa Cells
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A mixture of RIPA lysis buffer (Beyotime, China) and protease inhibitor (CMBIO, China) at a ratio of 100:1 was used to lyse BCa cells. Samples containing 30 μg of total protein were separated by 10% SDS–PAGE, and then the protein was transferred onto PVDF membranes for 2 hours. After blocking with 5% nonfat milk in TBST for 1 h at room temperature, anti-BARX2 (Bioss, China; cat. No. bs-19273R-3; dilution: 1:800), anti-c-MYC (Proteintech, China; cat. NO. 10828-1-AP; dilution: 1:5000), anti-β-catenin (Proteintech, China; cat. NO. 51067-2-AP; dilution: 1:5000), anti-GAPDH (Proteintech, China; cat. NO. 10494-1-AP; dilution: 1:10000), anti-Histone H3.1 (Beijing Ray Antibody Biotech, China; cat. NO. RM2005L; dilution 1:5000) and anti-β-actin (Proteintech, China; cat. No. 66009-1-Ig; dilution: 1:10000) antibodies were used to incubate PVDF membranes at 4° C overnight. The next day, the membranes were incubated with HRP-coagulated anti-rabbit or anti-mouse antibody at room temperature for 1 h.
6
Western Blot Analysis of EMT Markers
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Cells were lysed with RIPA lysate (Beyotime, Shanghai, China), and the concentration of protein was determined by the BCA assay kit (Solarbio, Beijing, China). Briefly, the equal amount of protein was separated with 10% SDS-PAGE (Epizyme, Shanghai, China), and transferred to PVDF membranes (Millipore, MA, USA). The PVDF membranes were blocked with 5% skim milk and incubated at 4 °C overnight with primary antibody, including anti-Fibronectin (Cell Signaling Technology, MA, USA), anti-N-cadherin (CST), anti-E-cadherin (CST), anti-MMP2 (CST), anti-MMP9 (CST), anti-Cytokeratin (Proteintech, Wuhan, China), anti-Vimentin (CST), anti-Ran (Proteintech), anti-Akt (CST), anti-p-Akt (CST), GSK3β (CST), anti-p-GSK3β (CST), anti-β-catenin (CST). β-actin (CST) was used as internal controls. Then the PVDF membranes were incubated with a secondary antibody for 1 h. The blots were scanned by Amersham Imager 600 (GE, Boston, MA, USA). Each sample was repeated three times and the three independent western blot bands were quantitatively analyzed by Image J software (NIH, Bethesda, Maryland, USA).
7
Protein Extraction and Western Blotting
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Proteins were extracted from cell lines or tissue and lysed in GLB+ supplemented with a protease inhibitor cocktail (Bimake) for 20 min on ice, followed by centrifugation (12,000 rpm, 4° C, 15 min). Membrane and cytoplasmic proteins were prepared with the Mem-PER™ Plus Membrane Protein Extraction Kit (Thermo, 89842). Then, 25-60 μg of protein extracts were loaded onto 8-12% electrophoresis gels and transferred to membranes. The membranes were blocked with 5% non-fat milk and subsequently incubated with primary antibodies. The antibodies used were anti-ANXA4 (Proteintech), anti-CAMK2G (Proteintech), anti-HA (Proteintech), anti-FLAG (Sigma), anti-E-cad (Cell Signaling Technology), anti-N-cad (Cell Signaling Technology), anti-Vimentin (Cell Signaling Technology), anti-Snail 1 (Proteintech), anti-β-catenin (Proteintech), anti-Tubulin (Proteintech), anti-GAPDH (Proteintech) and anti-Na+-K+-ATPase (Santa Cruz).
8
Immunofluorescence Staining of Cytoskeleton
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The cells seeded in co focal dishes were fixed with 4% paraformaldehyde and then kept stable in 0.2% Triton for 10 min to rupture the cell membranes. Following three PBS washing, non-specific antigen-binding sites were blocked by 2% BSA for 30 min. The cells were then incubated with anti- Vimentin, E-cadherin (Abcam, 1:200); anti-β-catenin (Proteintech, 1:50) overnight at 4 °C. After washing, cells were incubated with anti-rabbit antibody for 60 min and the nuclei were stained with DAPI for 2 min, which was washed with PBS later. Cells were kept from light before observed with a fluorescence microscope.
9
Immunofluorescence Staining of Cell Markers
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1 × 105 cells were seeded on cell climbing slices coated with poly-D-lysine in 24 well plates. Prepared cells were fixed in 4% PFA for 20 min, permeabilized with 0.5% Triton X-100 for 5 min, and blocked in 10% goat serum for 1 h at room temperature. The following primary antibodies were incubated overnight at 4 °C: anti-LDHA (1:250, Proteintech), anti-β-catenin (1:200, Proteintech), or anti-HIF-1α (1:200, Abcam; 1:100 Proteintech). Next day, cells were incubated with secondary antibodies for 1.5 h at room temperature: CoraLite594-conjugated goat anti-rabbit IgG(H + L) (1:250, Proteintech), CoraLite488-conjugated goat anti-mouse IgG(H + L) (1:250, Proteintech). Cell climbing slices were mounted on slides with mounting medium containing DAPI and photographed by a TCS SP8 confocal laser scanning microscopy (Leica, Italy) or an Axio Scope A1 microscope (Zeiss, Germany) at 400× magnification.
10
Immunohistochemical Analysis of Tumor Samples
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Tumors were fixed in 4% paraformaldehyde immediately after removal from nude mice. After tissue embedding, slicing, dewaxing, and incubating with antibodies, images were taken at the proper magnification using a microscope (Leica Microsystems, Mannheim, Germany). Antibodies used in this experiment were, anti-PCNA (10205-2-AP, Proteintech, USA), anti-SRSF1(sc-33652, Santa Cruz Biotechnology, China) and anti-β-catenin (51067-2-AP, Proteintech, USA).
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