Primary antibodies: β-catenin from BD-Pharmingen, San Jose, CA, USA (catalog #: 610156 for immunofluorescence); β-catenin from BD-Pharmingen (catalog #: 610153 for immunoblot); survivin from Santa Cruz, Dallas, TX, USA (catalog #: sc-10811), E-cadherin from Santa Cruz (catalog #: sc-7870), CBP from Santa Cruz (catalog #: sc-369), p300 from Santa Cruz (catalog #: sc-584); K-Ras from Santa Cruz (catalog #: sc-30), Lamin A/C from Santa Cruz (catalog #: sc-7293), α-tubulin from Calbiochem, Burlington, MA, USA (catalog #: CP06), EGFR from Santa Cruz (catalog #: sc-03). Secondary antibodies for immunofluorescence were purchased from Invitrogen (Waltham, MA, USA).
β catenin
Manufactured by BD
Sourced in United States, United Kingdom, Germany, Uruguay, China, Canada
β-catenin is a protein that plays a crucial role in cell-cell adhesion and the Wnt signaling pathway. It is involved in the regulation of gene expression, cell proliferation, and cell differentiation. β-catenin is a key component of the adherens junctions between cells, where it links cadherin cell adhesion molecules to the actin cytoskeleton.
Automatically generated - may contain errors
Lab products found in correlation
E cadherin, by BD (69 mentions)
β actin, by Merck Group (46 mentions)
N cadherin, by BD (33 mentions)
Flag tag (flag), by Merck Group (17 mentions)
Cleaved caspase 3, by Cell Signaling Technology (15 mentions)
β actin, by Santa Cruz Biotechnology (14 mentions)
β actin, by Cell Signaling Technology (13 mentions)
α tubulin, by Merck Group (13 mentions)
E cadherin, by Cell Signaling Technology (13 mentions)
Vimentin, by BD (13 mentions)
Gsk3β, by Cell Signaling Technology (13 mentions)
Cyclin d1, by Cell Signaling Technology (12 mentions)
Fibronectin, by BD (11 mentions)
Lysozyme, by Agilent Technologies (10 mentions)
Axin2, by Cell Signaling Technology (10 mentions)
C myc, by Cell Signaling Technology (9 mentions)
Gapdh, by Merck Group (9 mentions)
Vimentin, by Cell Signaling Technology (9 mentions)
Gapdh, by Santa Cruz Biotechnology (8 mentions)
Gapdh, by Cell Signaling Technology (8 mentions)
387 protocols using β catenin
1
Antibody Detection in Cell Signaling
2
Immunofluorescence and Immunoblotting for AhR and β-Catenin
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AhR and β-catenin localization changes were examined via immunofluorescence (IF) staining as described in a previous report [58 (link)]. Briefly, shCtrl and shIDO1 HuH-7 and Sk-Hep1 cells were treated with or without IFN-γ (40 ng/mL) in the absence or presence of KYN at the indicated doses for 48 h at 37 °C. After treatment, cells were fixed, blocked, and immunostained with antibodies against AhR (Santa Cruz, Dallas, TX, USA) and β-catenin (BD Biosciences, San Jose, CA, USA), followed by fluorescein isothiocyanate (FITC)-labeled (AhR) or tetramethylrhodamine-isothiocyanate (TRITC)-labeled (β-catenin) secondary antibodies. Nuclear staining was conducted with 4′,6-diamidino-2-phenylindole (DAPI) and images captured using an Olympus BX-51 microscope (Olympus, Japan). For immunoblot analysis, HuH-7, Sk-Hep1 and Ph5Ch8 cells were subjected to lentivirus-mediated knockdown of IDO1 or AhR and exposed to IFN-γ (40 ng/mL) or KYN at the indicated doses and times. All samples were lysed in RIPA containing 150 mM NaCl, 5 mM EDTA, 25 mM Tris-HCl, 1% NP 40, 0.1% SDS, 1× protease inhibitor cocktails, pH 8.0. Lysates were separated via SDS-PAGE and transferred to PVDF membranes. Antibodies used for subsequent immunoblotting are listed in Table S2 .
3
Western Blot Analysis of Protein Signaling
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Cellular protein was extracted with cell lysis buffer (CelLytic-MT, Sigma-Aldrich). A 30 μg aliquot of protein extract was analyzed by Western immunoblotting for the proteins of interest as described previously [15 (link), 16 (link)]. The amount of protein in each sample was normalized to the expression of β-actin. The following primary antibodies were used at the dilutions shown against the indicated proteins: both GSK-3 isoforms (GSK-3α and β) (1:1,000, Millipore, Billerica, MA); GSK-3β (1:1,000, BD Biosciences, Lexington, KY); GSK-3β fractions phosphorylated at the serine (S) 9 residue (pGSK-3βS9), (1:1,000, Cell Signaling Technology, Beverly, MA); and at the tyrosine (Y) 216 residue (pGSK-3βY216) (1:1,000, BD Biosciences); β-catenin (1:1,000; BD Biosciences); β-catenin phosphorylated at S33, S37 and/or threonine (T) 41 residues (p-β-cateninS33/37/T41) (Cell Signaling Technology); AKT (1:1,000) and its fractions phosphorylated at T308 residue (pAKTT308) (1:1,000) and at S473 residue (pAKTS473) (1:1,000) (Cell Signaling Technology); and β-actin (1:4,000, Ambion, Austin, TX).
4
Immunohistochemical and Western Blot Analysis
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β-Catenin (610154, BD); caspase-3 (9661L, Cell Signaling); cyclin D1 (2978S, Cell Signaling); lysozyme (A0099, Dako); Sox9 (AB5335, Millipore); Olfm4 (39141S, Cell Signaling); USP7 (Bethyl Laboratories); CD44 (MAB2137, Merck); and keratin 20 (13063S, Cell Signaling) were used in immunohistochemistry analysis. α-Actinin (sc-15335) and β-Catenin (610154, BD) were used for western blot analysis. DUB inhibitor VI P22077 (Calbiochem) was resuspended in DMSO at 10 mM (for organoid experiments) or 15 mg/mL (for mice experiments).
5
Liver Pathology Analysis via Immunohistochemistry
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Mice were sacrificed by carbon dioxide asphyxiation. Livers were fixed in 4% formalin overnight, embedded in paraffin, sectioned at 4 μm and stained with hematoxylin and eosin (H&E) for pathology. Liver sections were de-waxed, rehydrated and stained using standard immunohistochemistry protocols32 (link). The following antibodies were used: anti-Pten (Cell Signaling, 9559, 1:100), anti-pAkt S473 (Cell Signaling, 4060, 1:50), β-Catenin (BD,610154, 1:100), anti-p53 (CM5, 1:300), anti-GS (BD 610517, 1:200) and anti-Ck19 (Abcam, ab133496, 1:100). The number of hepatocytes was quantified from >3 low-magnification fields per mouse with 5 mice per group. Immunofluorescence was performed as previously described32 (link). β-Catenin (BD,610154) and phospho-β-Catenin (Abcam, ab53050) antibodies were used. Slides were counterstained with 4, 6-diamidino-2-phenylindole (DAPI). Images were obtained with a Nikon A1R laser scanning confocal microscope using a 40× APO Fluor objective (NA 0.65).
6
Immunoblotting and Immunofluorescence Assay Protocol
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Blebbistatin (B05060), low-endotoxin BSA (A8806), PA (P9767), and Y27632 (Y0503) were from Sigma Aldrich (St. Louis, MO), while cis-PO/palmitoleic acid (10009871) was from Cayman Chemicals (Ann Arbor, MI). Antibodies used for immunoblotting were α-actinin (Millipore Sigma; A7811), β-actin (Cell Signaling; 4790), α-catenin (ThermoFisher; 13-9700), β-catenin (BD Biosciences; 610153), VE-cadherin (Santa Cruz; 9989), RhoA (Santa Cruz; c-418), IRDye 800CW goat anti-rabbit immunoglobulin G (IgG) (LI-COR; 9253211), and IRDye 680LT goat anti-mouse IgG (92668070). Antibodies and reagents used for immunofluorescence were α-catenin (ThermoFisher; 13-9700), β-catenin (BD Biosciences; 610153), DAPI (4’,6-Diamidino-2-phenylindole, dihydrochloride; ThermoFisher; D1306), GM130 (BD Biosciences; 610823), phospho-MLC (Cell Signaling; 3674), phalloidin (Alexa Fluor 555) (ThermoFisher; A34055), phospho-YAP S127 (Abcam; 76252), YAP (Cell Signaling 14074), VE-cadherin (Santa Cruz; 9989), goat anti-rabbit IgG secondary antibody (Alexa Fluor 488) (ThermoFisher; A11008), goat anti-mouse IgG secondary antibody (Alexa Fluor 647) (ThermoFisher; A21235). Dako fluorescence mounting medium (S3023) was from Aligent. Y227632 (Y0503) was from Millipore Sigma (Burlington, MA). The plasmid encoding GFP–β-actin was kindly provided by Sergio Grinstein (Hospital for Sick Children, Toronto, Ontario, Canada).
7
Smad1 Regulation by miR-26a-5p in Osteoblast Differentiation
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Control siRNA and Smad1 siRNA were ordered from Sigma. The miR‐26a‐5p mimic and inhibitor were ordered from Dharmacon. Transfection was performed as previously described.16 Western blot analysis was performed as described previously,17 using antibodies against β‐actin (Sigma‐Aldrich), Smad1 (Cell Signaling), Lef1 (Cell Signaling), β‐catenin (Becton Dickinson) and active β‐catenin (EMD Millipore). Luciferase reporter assays were performed as previously described.16 Briefly, the sequences containing the putative binding sites 1 (5′‐atcgagccttgcatgTACTTGAA‐3′) and 2 (5′‐aaggagccacgataaTACTTGAc‐3′) in the 3′‐UTR of Smad1 were inserted into the cloning site of the CMV‐Luc2 reporter vector. To mutate the binding sites, the seed sequences (the capitalized parts in above sequences) for the miR‐26a‐5p‐Smad1 interaction were deleted. Control siRNA or miR‐26a‐5p mimic was cotransfected with CMV‐luc2 plasmids as well as Renilla luciferase plasmids into mouse CD45− bone marrow stromal cells. To induce osteoblast differentiation of disc cells, after reaching 100% confluency, cells were cultured in α‐MEM supplemented with 10% FBS, 10 nmol/L dexamethasone, 50 µg/mL ascorbic acid and 10 mmol/L β‐glycerophosphate. Alkaline phosphatase (ALP) staining was performed as previously described.18
8
Organoid Fractionation and Protein Analysis
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Organoids were lysed with RIPA buffer (Sigma-Aldrich, St. Louis, MO, USA) with EDTA-free protease inhibitor cocktail (Roche Applied Science, Mannheim, Germany). Nuclear and cytosolic protein was fractionated using Nuclear/Cytosol Fractionation Kit (BioVison). Subsequently, protein concentration was measured by BCA Protein Assay Kit (Thermo Fischer Scientific) and western blotting was performed as previously described [50 ]. Primary antibodies were used; GAPDH (sc32233, 1:1000, Santa Cruz Biotechnology, Santa Cruz, CA), β-catenin (610154, 1:2000, Becton Dickinson, Frankilin Lakes, NJ), FOXA1 (ab170933, 1:1000, Abcam, Cambridge, MA, USA), Cytokeratin 20 (CK20) (ab76126, 1:10000, Abcam), Cytokeratin 5 (CK5) (PRB-160P, 1:1000, Covance).
9
Immunofluorescence Assay for Wnt Pathway Components
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Cells for immunofluorescence were fixed with PBS-3.7% formalin for 20 min and cells were subsequently permeabilized with PBS-0.2% Triton X-100 solution for 10 min. Non-specific interactions were blocked with PBS-3% BSA. Primary and secondary antibody incubations were performed in PBS-3% BSA for 1 h at room temperature. Fluorescence detection was performed using Alexa-Fluor 488/594/647 (Invitrogen, USA) secondary antibodies and Hoechst dye (H6024, Sigma Aldrich, USA) nuclear stain. The following primary antibodies were used for western blotting and immunofluorescence: Axin (AF3287, R&D Systems, USA), axin (C76H11, 2087, CST, USA), β-catenin (610153, Becton Dickinson, USA), β-actin (A5316, Sigma-Aldrich, USA), TNKS (H-350) (sc-8337, SCBT, USA), GSK3β (L-17) (sc-8257, SCBT, USA), APC (H-290) (sc-7930, SCBT, USA), phosho β-catenin (9561, CST, USA), non-phosho β-catenin antibodies (8814, CST, USA) and α-tubulin (T6074, Sigma-Aldrich, USA).
Immunofluorescence microscopy cell imaging acquisition was performed with an Olympus DeltaVision core deconvolution microscope with SoftworxResolve 3D software. Images were taken for every field using a 40x objective and deconvolution was used to process the images.
Immunofluorescence microscopy cell imaging acquisition was performed with an Olympus DeltaVision core deconvolution microscope with SoftworxResolve 3D software. Images were taken for every field using a 40x objective and deconvolution was used to process the images.
10
Protein Extraction and Analysis from Cells
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Cells were lysed in Triton lysis buffer (TBS (50 mM Tris pH7.4, 150 mM NaCl, 2.7 mM KCl), 1% Triton X-100, 2 mM β-mercaptoethanol (ME), 1 mM MgCl2, 1 × proteinase inhibitor cocktail (PIC) (Sigma)) for 5 min on ice. Lysates were cleared by centrifugation and analyzed by SDS-PAGE. For β-catenin analysis, cytosolic extracts were prepared using Saponin lysis buffer (0.05% saponin, 1 mM MgCl2, 1 × TBS, 2 mM ME, 1 × PIC), for 30 min on ice. Antibodies used were: SOX4 (Diagenode cs-129-100), α-tubulin (Thermo Scientific, MS-581-P0), DDIT3 (Santa Cruz sc-575), β-catenin (BD Transduction Laboratories, 610153), and TCF4 (Santa Cruz, sc-166699).
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