Fifty micrograms of proteins extracted from the cell lysates was solubilized in SDS buffer and boiled for 5 min. Samples were separated on 12.5 % polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 5 % skim milk in Tris-buffered saline (TBS) at room temperature for 1 h or 4 °C overnight and incubated with a primary antibody for 1 h at room temperature. The blots were then probed with primary antibodies, including mouse anti-β-catenin antibody (1:1,000, BD Biosciences, San Jose, CA), mouse anti-cyclin D1 (1:1,000, Cell signaling, Danvers, MA), and mouse anti-β-actin (1:20,000, Sigma-Aldrich, St. Louis, MO, USA) as an internal control. After rinsing with TBS containing 0.1 % polyoxyethylene sorbitan monolaurate (Tween-20 or TBS-T), membranes were incubated with horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biochemical, Santa Cruz, CA) at room temperature for 1 h. After rinsing with TBS-T, membranes were exposed to the ECL Plus Western Blotting Detection System (GE Healthcare Bio-Science, UK) for 5 min. Human GAPDH was used as a loading control. The immunoblot and intensity were analyzed by an ImageQuant analysis system (GE Healthcare Bio-Science, UK).
Mouse anti β catenin antibody
Manufactured by BD
Sourced in United States
The mouse anti-β-catenin antibody is a primary antibody that specifically binds to and recognizes the β-catenin protein. β-catenin is a key regulator of the Wnt signaling pathway and plays a role in cell-cell adhesion.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Mouse anti cyclin d1, by Cell Signaling Technology (1 mentions)
Imagequant analysis system, by GE Healthcare (1 mentions)
Mouse anti β actin, by Merck Group (1 mentions)
Ecl plus western blotting detection system, by GE Healthcare (1 mentions)
Ripa buffer, by Boster Bio (1 mentions)
Mouse anti α tubulin antibody, by Merck Group (1 mentions)
Mouse anti vinculin antibody, by Merck Group (1 mentions)
Rabbit anti fak antibody, by Santa Cruz Biotechnology (1 mentions)
Chamber slide, by BD (1 mentions)
Mouse anti brdu antibody, by BD (1 mentions)
Mouse monoclonal anti cd133 w6b3c1 clone, by Miltenyi Biotec (1 mentions)
Rabbit anti sox2 antibody, by Merck Group (1 mentions)
Mouse anti nestin antibody, by Merck Group (1 mentions)
Rabbit anti olig2, by Abcam (1 mentions)
P smad2, by Abcam (1 mentions)
Tgfβr1, by Abcam (1 mentions)
Vectastain abc kit, by Vector Laboratories (1 mentions)
Alexa fluor 680 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
14 protocols using mouse anti β catenin antibody
1
Western Blot Analysis of Cell Signaling
2
Western Blot Analysis of ZBP1, β-Catenin, and Housekeeping Proteins
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The cells were rinsed with cold PBS and lysed in RIPA buffer (Boster Biological Technology, Wuhan, China) for 30 min with constant agitation. A 10–40 μg aliquot of the supernatant from each sample mixed with loading dye was boiled at 95 °C for 4 min and then separated on sodium dodecyl sulfate-polyacrylamide gels at 80–120 V. Western blotting was carried out as previously described.42 (link) The primary antibodies used included a rabbit anti-ZBP1 antibody (Invitrogen), a mouse anti-β-catenin antibody (BD Biosciences), a mouse anti-TATA-binding protein TBP antibody (Abcam), and a mouse anti-α-tubulin antibody (Sigma-Aldrich).
3
Western Blot Protocol for Cell Signaling
4
Auranofin Effects on β-catenin Localization
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The T41A-mutated cells in DMEM containing 10% FBS were seeded onto chamber slides (FALCON, Big Flats, NY) at 1.0 × 105 cells/ml and incubated for 12 h. The culture medium was replaced with 10% FBS-containing DMEM containing auranofin (dissolved in DMSO) at each 0–5 µM concentration and cultured for 24 h, followed by fixation with 4% paraformaldehyde for 2 h at room temperature. Cells were permeabilized in phosphate-buffered saline (PBS) containing 0.2% Triton X-100 by incubation at room temperature for 10 min. Preparations were incubated in blocking buffer containing 10% goat serum for 60 min at room temperature and incubated with mouse anti-β-catenin antibody (1:500 dilution, BD Biosciences, Franklin Lakes, NJ) at 4 °C overnight. Next, they were incubated with goat anti-mouse fluorescein isothiocyanate (FITC) secondary antibody (1:100 dilution, Proteintech) in a dark environment for 1 h at room temperature. Hoechst 33342 (5 μL/mL, Thermo Fisher Scientific) was added to them and incubated for 10 min at room temperature in a dark environment.
5
Antibody Profiling of Neural Stem Cells
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The antibodies used were as follows: mouse anti-β-catenin antibody, mouse anti-Stat3 antibody and mouse anti-BrdU antibody were from BD Biosciences; mouse anti-Bmi-1 antibody and rabbit anti-OLIG2 and rabbit anti-POU3F2 was from Abcam; mouse anti-PTEN antibody and rabbit anti-CHOP antibody were from Cell Signaling Technology; mouse anti-Nestin antibody and rabbit anti-Sox2 antibody were from Millipore, mouse monoclonal anti-CD133 (W6B3C1 clone) was from Miltenyi Biotec.
6
Kidney Immunohistochemistry Protocol
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Kidney sections (5 μm thick) were used for immunohistochemistry. To block endogenous peroxidase, all sections were incubated in 0.3% hydrogen peroxide for 10 minutes. Immunohistochemical staining was performed using a Vectastain ABC Kit (Vector Laboratories, Burlingame, CA). Rabbit polyclonal TGFβR1 (1:100) and rabbit polyclonal p‐smad2 (1:100) antibodies were purchased from Abcam (Cambridge, MA). Mouse anti–β‐catenin antibody (1:100) was purchased from BD Biosciences. In the negative controls, the primary antibody was omitted and replaced with the blocking solution. We analyzed 15 to 20 stained glomeruli from each mouse.
7
Immunofluorescence Localization of β-Catenin
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Cells were seeded on cover slips in six-well plates and allowed to adhere overnight until the cells reached 60%–70% confluence. The cells were subsequently treated with 0.5 μM GDC-0941 for 18 hr, followed by two washes with PBS buffer. Then, the cells were fixed using 4% paraformaldehyde for 25 min at room temperature, permeabilized with 1% Triton X-100 for 10 min, and incubated with a purified mouse anti-β-catenin antibody (BD Biosciences; 1: 500) at room temperature for 2 hr. The secondary antibody was an Alexa Fluor 680 goat anti-mouse IgG (Life Technologies). DAPI was used to visualize DNA. Fluorescence microscopy was performed in the City of Hope Microscope Core.
8
Protein Extraction and Western Blot Analysis
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Samples were lysed in protein lysis buffer which was composed of 50 mM Tris (pH 7.5), 100 mM NaCl, 1 mM EDTA, 0.5% NP40, 0.5% TritonX-100, 2.5 mM sodium orthovanadate, 10 μM protease inhibitor cocktail, and 1 mM phenylmethylsulfonyl fluoride. Extracted protein was separated by 10% SDS-PAGE, and transferred onto PVDF membranes (Millipore, Bedford, USA). The membranes were blocked in 5% milk in 1 × TBST for one hour at room temperature, followed by incubation with the respective primary antibodies at 4°C overnight. After washed with TBST, the membranes were incubated with horseradish peroxidase conjugated secondary antibodies (1:5000; CST, USA). An enhanced chemiluminescence (ECL) kit (Millipore, Bedford, USA) was used to visualize protein bands on PVDF membranes. Primary antibodies used in this study were listed as the following: mouse anti-GAPDH antibody (1:3000, Kangcheng, Shanghai, China), rabbit anti-PDE4D antibody (1:1000, Proteintech, USA), mouse anti-β-Catenin antibody (1:1000, BD, USA), mouse anti-N-cadherin antibody and rabbit anti-Snail (1:1000, CST, USA).
9
Western Blot Analysis of β-Catenin
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Cell lysates were prepared by washing the cells with PBS twice followed by the addition of MPER mammalian extraction reagent (Pierce). An aliquot of the protein was added to 2X SDS loading buffer consisting of 4% v/v SDS, 200 mM dithiothreitol, 100 mM Tris pH 6.8, 20% v/v glycerol, 0.2% w/v bromphenol blue for polyacrylamide gel electrophoresis (PAGE). Protein concentration was measured using the Bio-Rad protein assay on the lysate samples, with known amounts of bovine serum albumin to establish a standard curve. Approximately 10 μg of protein from each experimental sample group was loaded onto a 4%-20% Tris-glycine SDS-PAGE gel. The size-resolved proteins were transferred to Immobilon-P membranes (Millipore) for 1 hour at 100 mA. The membrane was blocked with 5% non-fat milk in TBST (1xTBS, 0.1% Tween-20) for 1 hour at room temperature. Mouse anti-β-catenin antibody (1:2000; BD Bioscience) was added to the TBST and the membrane was incubated at 4°C overnight. HRP-conjugated antibody was used as a secondary antibody and incubated with the membrane for 1h. The antigen-antibody signal was detected by ECL detection system and normalized to the amount of β-actin from the same sample. Quantification of the signal was performed as described previously [4] .
10
Quantifying Beta-catenin Expression in Growth Plates
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OAC were fixed with 4% paraformaldehyde at room temperature. Coronal paraffin sections of proximal tibial growth plates were deparaffinized and hydrated in xylene. The specimens were then treated with a blocking buffer including 2% goat serum in 0.5% Triton-X100 for 60 minutes and incubated with mouse anti-β-catenin antibody (BD Transduction Laboratories, 1∶500 dilutions for the cells and 1∶100 dilutions for the sections) at 4 °C overnight. The specimens were incubated with goat anti-mouse fluorescein isothiocyanate (FITC) secondary antibody (1: 500 dilution) at room temperature for one hour. Finally, the specimens were mounted in VectaShield containing 1.5 μg/ml DAPI (Vector Laboratories, Peter-borough, UK) and visualized using IX71 (Olympus). The ratio of β-catenin-positive cells were automatically estimated by dividing FITC-positive cells by the number of DAPI-positive nuclei using MetaMorph (Molecular Device)
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