MCF-7 and MDA-MB-231 cells were cultured in 35-mm plates with cover slides on the plate bottom and treated with NLS-BLBD-6, TAT-BLBD-6 and TAT-NLS-BLBD-6 peptide for 24 hr. Subsequently, cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100 for 20 min at room temperature. For the immunofluorescence assay, the slides were incubated with primary antibodies, anti-β-catenin (Epitomics) and anti-TAT (Santa Cruz), for 6 hr, and Alexa-488 fluorescence secondary antibodies for 1 hr. DAPI (Sigma, St Louis, MO) was used to stain the nucleus for 1 min at room temperature. For the TUNEL assay, apoptosis was analyzed by the Apo-BrdU-red DNA Fragmentation Assay kit, according to the manufacturer’s protocol (BioVision, Mountain View, CA, USA). For the PLA, the protein–protein interaction assay was analyzed by Duolink® using PLA® Technology, according to the manufacturer’s protocol (Olink Bioscience, Uppsala, Sweden). Briefly, the slides were incubated with primary antibodies, anti-β-catenin (Epitomics) and anti-TAT (Santa Cruz), and secondary antibodies, Duolink PLA Rabbit MINUS and PLA Mouse PLUS proximity probes. Finally, the proximity ligation was performed by the Duolink detection reagent kit (Olink Bioscience). The immunofluorescence image and TUNEL staining was photographed by a microscope (IX-71, Olympus, Tokyo, Japan).
Anti β catenin
Manufactured by Abcam
Sourced in United States, United Kingdom, China
Anti-β-catenin is a primary antibody that specifically binds to the β-catenin protein. β-catenin is a key component of the Wnt signaling pathway and plays a role in cell-cell adhesion and gene transcription.
Automatically generated - may contain errors
Lab products found in correlation
Anti gapdh, by Abcam (28 mentions)
Pvdf membrane, by Merck Group (24 mentions)
Anti c myc, by Abcam (23 mentions)
Anti cyclin d1, by Abcam (23 mentions)
Anti β actin, by Abcam (22 mentions)
Anti e cadherin, by Abcam (21 mentions)
Anti vimentin, by Abcam (20 mentions)
Anti gsk3β, by Abcam (20 mentions)
Anti n cadherin, by Abcam (17 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (12 mentions)
Anti bax, by Abcam (10 mentions)
Anti bcl 2, by Abcam (10 mentions)
Ripa lysis buffer, by Beyotime (9 mentions)
Anti dkk1, by Abcam (9 mentions)
Anti e cadherin, by Cell Signaling Technology (8 mentions)
Ripa buffer, by Beyotime (8 mentions)
Anti p gsk 3β, by Abcam (8 mentions)
Anti gapdh, by Cell Signaling Technology (8 mentions)
Anti gapdh, by Santa Cruz Biotechnology (7 mentions)
Anti akt, by Abcam (7 mentions)
228 protocols using anti β catenin
1
Visualizing Protein-Protein Interactions in Cancer Cells
2
Immunoprecipitation of β-catenin
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In brief, cold phosphate-buffered saline) was used to wash the cells three times, after which the cells were scraped into lysis buffer containing complete protease inhibitors and were centrifuged at 14,000 × g for 20 min at 4 °C. The supernatants were incubated with anti-β-catenin (Abcam; 1:1000) or normal IgG (Abcam; 1:1000) antibody, and the immunocomplexes were then associated with protein A-sepharose. anti-β-catenin (Abcam; 1:1000) and anti-ubiquitin (Abcam; 1:1000) antibodies were used for the western blot analysis.
3
Immunofluorescence Staining of EMT Markers
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Cells were cultured on glass cover slips and fixed in 4% paraformaldehyde for 30 min. The cells were then permeabilized using 0.25% Triton X-100 for 10 min at room temperature and blocked with 5% bovine serum albumin for 30 min at 37°C. The primary antibodies anti-EFEMP1 (1:200; Abcam, HK), anti-E-cadherin (1:100; CST, USA), anti-Vimentin (1:100; CST), anti-Snail (1:100; CST) and anti-β-catenin (1:100; Epitomics, Burlingame, CA, USA) were incubated with the samples overnight at 4°C. A species-specific secondary rhodamine-labeled antibody (1:200, Epitomics, USA) was incubated with the samples for 30 min at 37°C, and the cells were then counterstained with DAPI at room temperature for 10 min. Control samples were incubated with PBS in place of the primary antibody. Samples were mounted with antifade solution (Tocris; Ellisville, MO) prior to imaging using a laser scanning confocal microscope (Leica TCS-SP5 II, Heidelberg, Germany).
4
Investigating Wnt/β-catenin Pathway Regulation
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Rhein was purchased from MUST Biotech (Chengdu, China). LiCl was purchased from Sigma‐Aldrich (St. Louis, MO, USA). MG132 was from Merk Millipore Corporation (Darmstadt, Germany). Cycloheximide (CHX) was from Beyotime Biotechnology (Jiangsu, China). Anti‐β‐catenin, anti‐c‐Myc, anti‐PCNA, anti‐phosphorylated GSK‐3β (Ser9) antibodies were from Epitomics (Burlingame, CA, USA). Anti‐GSK3 antibody was from Signal way Antibody LLC (College Park, MD, USA). Anti‐cyclin‐D1 antibody was from Proteintech (Rosemont, IL, USA). The plasmid for phosphorylation‐deficient mutant of β‐catenin (S33A, S37A, T41A, S45A) was from Addgene (Cambridge, MA, USA).
5
Immunofluorescence and Immunoblotting Analysis
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Cells were cultured at a density of 1.5 × 105 cells/well on 8 mm coverslips in 12-well plates. After 48 hours, coverslips were fixed by ice-cold methanol, and incubated with primary E-cadherin (Abcam), Vimentin and β-catenin (Epitomics) antibodies prior to florescent-labeled secondary antibodies. Nuclear DNA was stained with 4′, 6-diamidino-2-phenylindole (DAPI) and coverslips were mounted with FluorSave reagent (CALBIOCHEM). Immunofluorescence images were taken by Olympus inverted fluorescence microscope and were outputted by PV10-ASW 1.7 viewer software. Immunoblotting was performed as follows: Proteins were extracted with lysis buffer and then quantified by the BCA method (KeyGen Biotech). Lysates were diluted in SDS sample buffer (KeyGen Biotech) prior to SDS-PAGE, and then transferred to a polyvinylidene difluoride membrane (Roche Applied Sciences). Membranes were immunoblotted overnight at 4°C with anti-SOX17 (Millipore), anti-CyclinD1 (Abclonal), anti-C-myc, anti-DKK1 (Cell Signaling Technology), anti-E-cadherin (Abcam), anti-SOX2, anti-β-catenin, anti-Vimentin and anti-Slug and anti-N-cadherin antibodies (Epitomics), followed by the appropriate second antibodies. The bands were exposed using Pierce ECL Western Blotting Substrate (Thermo Scientific). Gel densitometry (Bio-Rad) was used to quantify immunoblot signals on exposed film.
6
Evaluating β-Catenin Signaling in Breast Cancer
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Immunoprecipitation and western blotting were performed as described previously42 (link)43 (link). MCF-7 and MDA-MB-231 cells were harvested in 4 °C phosphate-buffered saline and cell pellets were lysed with RIPA lysis buffer (Millipore, Bedford, MA, USA) for 30 min on ice. Cell lysis supernatant liquid was obtained by centrifugation at 10,000 × g for 10 min, incubated with protein-G beads (Roche, Indianapolis, IN) and anti-β-catenin, and subjected to western blotting. For the western blotting assay, cellular extract proteins were separated by SDS-polyacrylamide gel (SDS-PAGE) and transferred to nitrocellulose membrane (Millipore) using a dry transfer apparatus (Bio-Rad). After blocking nonspecific binding with 5% milk buffer, the membrane was incubated with primary antibodies: anti-TAT (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-LEF-1(Epitomics, Burlingame, CA, USA) and anti-β-catenin (Epitomics, Burlingame, CA, USA). The proteins were visualized using ECL (Amersham Pharmacia Biotech) and coupled using the Bio-Rad Chemiluminescent Detection System.
7
Molecular Profiling of Epithelial-Mesenchymal Transition
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Experimental cancer cells (after culturing in HUVEC-CM for 21 days) and respective control cells were lysed on ice in a lysis buffer (RIPA, Beyotime, Shanghai, China) with a protease inhibitor mixture cocktail (Roche, Switzerland). Western-blot analysis was performed by established protocols 12 . Anti-α-catenin, anti-E-cadherin, anti-ZEB-1, ZEB-2, Snail, Slug, anti-ZO-1 and anti-Laminin A1 primary antibodies were purchased from (Abcam); anti-MMP-1, -2, -3, -11, -12, -13, -17 and -21, anti- ITGA6, B1, B3, B4, B7, anti-FAK, P-FAK-Y397, anti-Src, P-Src-Y418, P-Src-Y529, anti-fibronectin, anti-Laminin B3, anti-Wnt-2, anti-Wnt-5B, anti-Wnt-16, anti-TGF-β, anti-β-catenin and anti-N-cadherin primary antibodies were purchased from (Epitomics); anti-Cdc-2, P-Cdc-2-Tyr15, CDK4, CDK6, Cyclin A, Cyclin D1, Cyclin D3, Cyclin E2, P15, P16, P21, P27, P53, Rb, P-Rb-S811, P-Rb-S780; anti-Bcl-xl, Bad, P-Bad-Ser112, Bak, Mcl-1, Puma and anti-β-actin were from (Cell Signaling Technology).
8
Immunofluorescence Analysis of Cell Signaling
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Immunofluorescence was performed as described previously [17 (link)]. The following primary antibodies were used: anti-cytochrome c ( sc13561, Santa Cruz Biotechnology), anti-NFκB p65 (GTX102090, GeneTex), anti-β-catenin (E247, Epitomics #1247-s), anti-E-cadherin (GTX100443, GeneTex), anti-N-cadherin (TA326835, OriGene), anti-PTEN (#9188, Cell Signaling), anti-phospho-ERK1/2 (#4370, Cell Signaling) and anti-STAT3 (#12640, Cell Signaling). Goat anti-rabbit secondary antibody was coupled to AlexaFluor 555 (Invitrogen). Images were obtained with DS-Ri1 Nikon camera and Eclipse80i Nikon microscope and quantifications were performed using NIS-Elements BR 4.20.00 software (Nikon).
9
Immunofluorescence Analysis of Cell-Cell Adhesion
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The indicated cells were seeded onto coverslips in 6-well plates and cultured until 70% confluence. Cells were then fixed with 4.0% formaldehyde in phosphate-buffered saline for 15 min, and blocked with 5% goat serum for 30 min. Next, the coverslips were incubated at 4°C with primary antibodies overnight. Anti-E-cadherin, anti-Vimentin and anti-β-catenin antibodies were purchased from Epitomics, Inc. Subsequently, the coverslips were incubated with Cy3-conjugated goat anti-rabbit secondary antibody (Bioss, Beijing, P.R. China) and dried, dyed with Hoechst33342, and fixed in glycerol. The images were obtained with an Olympus IX71 microscope (Olympus, Tokyo, Japan), and color mergence was performed using ImageJ image software (ImageJ version 1.44p, NIH, MD).
10
Quantitative Protein Expression Analysis
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The total protein was extracted from the liver tissues using a Total Protein Extraction kit (Beyotime, Shanghai, China) and quantified using a BCA kit (Beyotime, Shanghai, China). Equal amounts of protein per sample (30 μg) were separated on a 10% SDS–PAGE and electroblotted onto nitrocellulose membranes (Pall Corporation, Port Washington, NY, USA). After blocking with 5% non-fat milk for 2 h, the blots were incubated overnight with anti-VEGF (1:2000; Wanleibio, Shenyang, Liaoning, China), anti-Cyclin D1 (1:1000; Wanleibio, Shenyang, Liaoning, China), anti-NLRP3 (1:2000; Bioss, Beijing, China), anti-Wnt2 (1:1000; Abcam, Waltham, MA, USA), anti-β-catenin (1:10,000; Abcam, Waltham, MA, USA), anti-P-P65 (1:500; Santa, Dallas, TX, USA), anti-P65 (1:3000; Proteintech, Wuhan, Hubei, China), anti-caspase-1 (1:500; Santa, Dallas, TX, USA), anti-ASC (1:500; Santa, Dallas, TX, USA), anti-GSDMD (1:2000; Affinity, Liyang, Jiangsu, China), and anti-β-tubulin (1:1000; Wanleibio, Shenyang, Liaoning, China) primary antibodies at 4 ℃. The blots were washed with TBST and incubated with HRP-conjugated anti-IgG for 2 h. The positive bands were detected using an enhanced ECL reagent (Meilunbio, Dalian, Liaoning, China) on an AI600 System (GE Healthcare, Pollards Wood, UK). The relative protein expression was quantified using ImageJ software and normalized against the β-tubulin control.
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