The following substances were obtained: doxorubicin, paclitaxel, G418, anti-HA (H6908), anti-CerS2 (HPA027262), anti-CerS4 (SAB4301210), anti-active-β-catenin (05-665), and anti-α-tubulin antibodies (T9026) (Sigma-Aldrich, St. Louis, MO, USA); anti-phospho-NF-κB p65 (3033), anti-NF-κB p65 (8242), anti-Relb (4922), anti-phospho-p44/42 MAPK (ERK1/2) (4370), anti-phospho-p90RSK (11989), anti-phospho-Akt (9271), anti-phospho-mTOR (5536), anti-phospho-p70 S6 kinase (9205), anti-ERα (8644), anti-phospho-ERα (Ser118) (2511), anti-phospho-ERα (Ser167) (64508), anti-PGR (8757), anti-vimentin (5741), anti-Snail (3879), anti-lamin A/C (2032), β-catenin (9562), and anti-phospho-GSK3β (5558) antibodies (Cell Signaling Biotechnology, Inc, Beverly, MA, USA); anti-LASS6 (CerS6) (sc-100554), anti-E-cadherin (sc-71008), and anti-N-cadherin (sc-59987) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-CerS1 antibody (H00010715-M01) (Abnova, Taipei, Taiwan); anti-GAPDH (glyceraldehyde 3-phosphate dehydrogenase) antibody (MAB374) (EMD Millipore, Billerica, MA, USA).
Anti active β catenin
Manufactured by Merck Group
Sourced in United States, Germany
Anti-active β-catenin is a laboratory reagent used in research applications. It is an antibody that specifically binds to the active form of the β-catenin protein, which is a key component of the Wnt signaling pathway. This product can be used to detect and quantify the levels of active β-catenin in biological samples, such as cell lysates or tissue extracts.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by Merck Group (6 mentions)
Anti β catenin, by BD (5 mentions)
Anti c myc, by Cell Signaling Technology (4 mentions)
Anti gapdh, by Cell Signaling Technology (3 mentions)
Anti β catenin, by Santa Cruz Biotechnology (3 mentions)
Anti β catenin, by Cell Signaling Technology (3 mentions)
Anti akt, by Cell Signaling Technology (2 mentions)
Anti rabbit igg whole molecule peroxidase, by Merck Group (2 mentions)
Anti phospho gsk3α β ser21 9, by Cell Signaling Technology (2 mentions)
Anti mouse igg whole molecule peroxidase, by Merck Group (2 mentions)
Anti phospho gsk 3α β y279 y216, by Abcam (2 mentions)
Ab15251, by Abcam (2 mentions)
Anti flag, by Merck Group (2 mentions)
Anti gapdh, by Santa Cruz Biotechnology (2 mentions)
Anti α tubulin, by Merck Group (2 mentions)
Anti β actin, by Cell Signaling Technology (2 mentions)
Ripa buffer, by Cell Signaling Technology (2 mentions)
Coomassie blue assay, by Thermo Fisher Scientific (2 mentions)
Anti β actin, by Santa Cruz Biotechnology (2 mentions)
Lsm 510 meta, by Zeiss (2 mentions)
39 protocols using anti active β catenin
1
Comprehensive Antibody Panel for Cell Signaling
2
Western Blot Analysis of Wnt Signaling
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Total proteins were extracted using RIPA buffer (NCM Biotech) with freshly added proteinase inhibitor. Proteins were then separated by 10–12% SDS/PAGE and transferred to 0.22 μm PVDF membranes (Millipore). The membranes were blocked with 5% skim milk and then incubated with primary antibodies overnight at 4 ºC. The primary antibodies used in this study were anti-PRICKLE1 (Proteintech, USA, 22589–1-AP) used at 1:1000, anti-DVL2 (Affinity, USA, DF4454) used at 1:1000, anti-LEF1 (Affinity, USA, DF7570) used at 1:1000, anti-Active β-catenin (Sigma-Aldrich, USA, 05-665) used at 1:1000, or anti-β-actin (Affinity, USA, DF7018) used at 1:1500. Appropriate HRP-conjugated secondary antibodies, and protein signals were developed with the enhanced chemiluminescence (ECL) reagents (Affinity Biosciences). ChemiDox XRS Chemiluminescence imaging system (Bio-Rad, USA) was used to capture and analyze the developed images.
3
Immunofluorescence Staining of Cell Markers
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The cells were fixed in 4% paraformaldehyde at 25 °C for 15 min. After washing with phosphate-buffered saline (PBS), the cells were blocked with blocking buffer (5% goat serum and 0.3% Triton X-100 in PBS) at room temperature for 1 h. After removing the buffer, the cells were incubated with primary antibodies (anti-Ki67, #12202; anti-active-β-catenin, #05-665, Merck Millipore, Billerica, MA, USA; and anti-TOMM20, ab186735, Abcam, Cambridge, UK) at 4 °C overnight. After washing with PBS, the cells were incubated with secondary antibodies (Alexa Fluor 555 anti-rabbit IgG, Alexa Fluor 488 anti-rabbit IgG, or Alexa Fluor 555 anti-mouse IgG) at 25 °C for 2 h. Then, the cells were washed and stained with Hoechst 33342 at room temperature for 20 min and observed under a fluorescence microscope (BZ-X800, Keyence, Osaka, Japan) [3 (link)].
4
Western Blot Analysis of Cellular Signaling
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Western blot analysis was performed as described previously (Zhou et al., 2019 (link)). Primary antibodies included rabbit polyclonal anti-fibronectin (F3648; Sigma-Aldrich), mouse monoclonal anti-β-catenin antibody (610154; BD Transduction Laboratories), mouse monoclonal anti-α-SMA antibody (A2547; Sigma-Aldrich), rabbit polyclonal anti-Wnt1 (ab15251; Abcam), mouse anti-α-tubulin (T9026; Sigma-Aldrich), mouse anti-PAI-1 antibody (AF3828;R&D Systems), anti-active β-catenin (#05–665; EMD Millipore), anti-Fas ligand (FasL) (SC -6237; Santa Cruz, CA, United States), p53 (#2524S; CST), Bax (SC-20067; Santa Cruz), p65 (#8242S; CST), p-p65 (#3033S; CST), PCNA (#2586S; CST).
5
Western Blot Analysis of Protein Markers
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Cultured cells or mouse livers were solubilized as described previously21 (link). The protein extracts were subjected to SDS-PAGE electrophoresis and immunoblotted with the following antibodies: anti-pAkt (pS473, Cell Signaling #4060, Boston, MA, USA), anti-Akt (Cell Signaling #9272), anti-β-actin (Sigma-Aldrich #A5316), anti-β-catenin (BD Transduction Laboratories #610153, San Jose, CA, USA), anti-active-β-catenin (Merck-Millipore #05-665, NJ, USA), anti-Glutamine Synthetase (BD Transduction Laboratories #610517), anti-IRβ (Santa Cruz, SC-711, Heidelberg, Germany), anti-p-S6K (pThr 389, Cell Signaling #9234), anti-SCD1 (Cell Signaling #2794). The FAS antibody was a kind gift from Dr. I. Dugail (UMR-ICAN, Paris, France). The immunoreactive bands were revealed using the ECL detection kit (Pierce ECL Western Blotting substrate, Rockford, IL USA). Autoradiograms were quantified using an imageJ program (Chemi Genius2 scan, GeneSnap; Syngene, Cambridge, UK).
6
EGF Regulation of β-Catenin Activation
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Primary MSCs were seeded at 2 × 104 cells per cm2 onto glass coverslips and left to adhere overnight. Cells were treated with varying concentrations of EGF (1 nM, 10 nM and 100 nM) and left for 24 h. Samples were fixed for 15 min in 4% paraformaldehyde before incubation with anti-active β-catenin (1:200, Merck) for 1 h at room temperature. Secondary antibody (Alexor Fluor 647, Invitrogen) was added for a further hour in the dark at room temperature. Samples were counterstained with DAPI, and mounted using Prolong Gold Antifade mountant before visualisation using the LSM710 confocal microscopy system.
7
Protein Expression Analysis Protocol
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To determine protein expression, cells were harvested in RIPA buffer containing a protease inhibitor cocktail, and total protein was quantified using a bicinchoninic acid kit (Pierce, Rockford, IL, USA). Aliquots containing 8 μg total protein were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and then electro-blotted onto a 0.45-μm PV membrane (Immobilon™; Merck Millipore, Darmstadt, Germany). The membranes were blocked and probed overnight with the primary antibodies anti-SEPP1 (1:1000, #ab193193; Abcam, USA), anti-MyoD (1: 1500; Invitrogen, Carlsbad, CA, USA), anti-MyoG (1: 1500; Invitrogen, Carlsbad, CA, USA), anti–β-catenin (1:5000, #ab32572; Abcam, USA), and anti–active β-catenin (1:500, #05-665; Merck Millipore).
8
Quantifying Active β-Catenin Levels
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For immunoblot, protein were run on a SDS-PAGE gel and transferred onto PVDF membranes, which were then incubated with specific primary antibodies followed by secondary antibodies for detection. Anti-Active-β-catenin was purchased from EMD Millipore (Billerica, MA). The epitope corresponds to amino acid residues 36–44 of β-catenin antibody, and specifically recognizes active form of β-catenin with dephosphorylation on Ser37 or Thr41.
9
Immunohistochemical Analysis of Wnt Signaling
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HCC tissues or orthotopic tumors were sectioned as 4‐μm‐thick slices and were dewaxed in xylene and rehydrated using 95% ethanol. The endogenous peroxidase activity was quenched by immersing the slides in a 0.3% H2O2 solution for 30 minutes at room temperature. Sections were incubated with primary antibodies overnight at 4°C. The horseradish peroxidase (HRP)‐conjugated secondary antibody was applied at 37°C for 60 minutes and incubated with diaminobenzidine solution, and nuclei were then counterstained with Harris’ hematoxylin. The relevant primary antibodies we used included anti‐Wnt8B (Abcam), anti‐ZNF191 (Abcam), anti–β‐catenin (Santa Cruz Biotechnology), anti–active‐β‐catenin (Merck Millipore), anti–Cyclin D1 (Santa Cruz Biotechnology), anti–c‐Myc (Cell Signaling), and Ki‐67 (Abcam).
The stained tissue slices were scored by two different pathologists blinded to patients’ clinical characteristics. The intensity of IHC staining was scored as 0 (negative), 1 (weak), 2 (medium), and 3 (strong). The percentage of positive cells in the whole tissue slice was recorded. Intensity score and positive rate score were then multiplied to calculate the overall score.
The stained tissue slices were scored by two different pathologists blinded to patients’ clinical characteristics. The intensity of IHC staining was scored as 0 (negative), 1 (weak), 2 (medium), and 3 (strong). The percentage of positive cells in the whole tissue slice was recorded. Intensity score and positive rate score were then multiplied to calculate the overall score.
10
Antibody Optimization for Protein Analysis
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Anti-phospho-GSK-3α/β (Ser21/9) (#9331), anti-GAPDH (#2118), and β-tubulin (#2146) were purchased from Cell Signaling Technology (Danvers, MA, USA). Both antibodies were used in a 1:1,000 dilution in TBS containing 3 or 5% BSA (PAA Laboratories, Pasching, Austria). Anti-phospho-GSK-3α/β (Y279/Y216) (#ab68476) and anti-WNT11 (#ab96730) were obtained from Abcam (Cambridge, UK) and used in a 1:1,000 and 1:3,000 dilution in TBS containing 3 or 5% BSA, too. Anti-active-β-catenin (#05-665) was purchased from Merck Millipore (Billerica, MA, USA) and used in a 1:2,000 dilution in TBS and 3 or 5% BSA (Carl Roth, Karlsruhe, Germany).
Anti-Akt (#9272), Anti-Akt (Ser473) (#9271), and Anti-Akt (Thr308) (#9275) were purchased from Cell Signaling (Billerica, MA, USA) and used in a 1:1,000 dilution in TBS and 3% BSA (Carl Roth, Karlsruhe, Germany). The species specific secondary antibodies anti-mouse IgG (whole molecule)-peroxidase (#A9044) and anti-rabbit IgG (whole molecule)-peroxidase (#A0545) were obtained from Sigma-Aldrich (St. Louis, MO, USA) and used in a 1:50,000 dilution in TBS containing either 5% BSA or 5% dry milk. Dead and apoptotic cells in response to 1,8-cineol were analyzed by annexin V and propidium iodide staining (Apoptosis Kit II; BD, Heidelberg, Germany).
Anti-Akt (#9272), Anti-Akt (Ser473) (#9271), and Anti-Akt (Thr308) (#9275) were purchased from Cell Signaling (Billerica, MA, USA) and used in a 1:1,000 dilution in TBS and 3% BSA (Carl Roth, Karlsruhe, Germany). The species specific secondary antibodies anti-mouse IgG (whole molecule)-peroxidase (#A9044) and anti-rabbit IgG (whole molecule)-peroxidase (#A0545) were obtained from Sigma-Aldrich (St. Louis, MO, USA) and used in a 1:50,000 dilution in TBS containing either 5% BSA or 5% dry milk. Dead and apoptotic cells in response to 1,8-cineol were analyzed by annexin V and propidium iodide staining (Apoptosis Kit II; BD, Heidelberg, Germany).
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