Cells were fixed and crosslinked in 1% formaldehyde, and lysed using RIPA buffer supplemented with protease-inhibitor cocktail and RNase inhibitor. The cell lysates were incubated with magnetic beads conjugated with anti-β-catenin antibody (Millipore). Mouse IgG (Millipore) was used as the negative control. The immunoprecipitated RNA was extracted from the eluate, and the enrichment of LINC01278 or control ACTB was analyzed by quantitative RT-PCR.
Anti β catenin antibody
Manufactured by Merck Group
Sourced in United States
The Anti-β-catenin antibody is a laboratory reagent used in Western blotting, immunohistochemistry, and other research applications. It specifically recognizes and binds to the β-catenin protein, which is a key component of the Wnt signaling pathway and plays a role in cell-cell adhesion and gene transcription. The antibody can be used to detect and quantify the expression of β-catenin in various biological samples.
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Lab products found in correlation
Anti his antibody, by Santa Cruz Biotechnology (1 mentions)
Mm00801666 g1, by Thermo Fisher Scientific (1 mentions)
Anti active β catenin antibody, by Merck Group (1 mentions)
Tri reagent, by Molecular Research Center (1 mentions)
Annexin 5 fitc apoptosis detection kit, by Immunotools (1 mentions)
Cyclosporine a, by Novartis (1 mentions)
Ldh cytotoxicity assay kit, by Abcam (1 mentions)
Amaxa cell line nucleofector kit 5, by Lonza (1 mentions)
Annexin 5 fitc apoptosis detection kit, by Abcam (1 mentions)
High glucose dulbecco s modified eagle s medium dmem, by Euroclone (1 mentions)
Anti flag antibody, by Merck Group (1 mentions)
Rpmi 1640, by Merck Group (1 mentions)
Luciferase assay system, by Promega (1 mentions)
Immobilon membrane, by Merck Group (1 mentions)
Nuclear extract kit, by Active Motif (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Pyrvinium pamoate, by Merck Group (1 mentions)
Anti lamin b1, by Abcam (1 mentions)
Anti gcsf antibody, by R&D Systems (1 mentions)
Anti c ebpα, by Abcam (1 mentions)
11 protocols using anti β catenin antibody
1
LINC01278 Immunoprecipitation Assay
2
Fracture Callus RNA and Protein Expression
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Total RNA or protein was extracted from fracture calluses after dissection. The proximal and distal borders of the fracture callus are roughly outlined by dashed lines in the low-magnification histological images. For real-time PCR, cDNA template was generated using random hexamers and data were related to the transcript of ribosomal protein 18S as a housekeeping control. Primers for Alp (Mm00475834_m1), BSP (Mm00492555_m1) and Col1 (Mm00801666_g1) were purchased from Applied Biosystems. For western blot analysis, indicated antibodies were detected using horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence (ECL). Relative band intensities were quantified using the ImageJ software ( http://imagej.nih.gov/ij/ ). Anti-β-catenin antibody (working concentration of 1.0 μg ml−l—06-734) was purchased from Millipore, anti-active β-catenin antibody (working concentration of 1.5 μg ml−1—05-665) was purchased from Millipore, anti-β-actin antibody (working concentration of 0.5 ng ml−1—CP01) was purchased from Calbiochem and anti-His antibody (working concentration of 0.2 ng ml−1—s-804) was purchased from Santa Cruz. Full western blot images are shown in Supplementary Fig. 12 .
3
Recombinant LMW HA and Doxorubicin Treatment
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Recombinant LMW HA (1–3 × 105 CPN Czech Republic) was kindly supplied by Farmatrade (Argentina). High glucose Dulbecco’s modified Eagle’s medium (DMEM) and DMEM F12 were purchased from MICROVET Laboratories (Argentina). TriReagent was from Molecular Research Center, Inc (USA). Doxorubicin (DOX) was kindly provided by Filaxis Pharmaceuticals S.A (Argentina). Anti-β-catenin antibody was purchased from Millipore (USA). Specific antibody against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was from NeoBioLab (USA). Anti-phosphorylated Akt (Ser473, Ser472 and Ser474) antibody was purchased from R&D System (USA) and anti-rabbit secondary horseradish peroxidase (HRP) antibody was from Santa Cruz Biotechnology (USA). CD44-APC antibody was from BD BioSciences (USA) and HA-FITC from Calbiochem (USA). Annexin V-FITC apoptosis detection kit was from ImmunoTools (Germany). Cyclosporine A was kindly provided by Novartis Pharmaceuticals Corporation (Argentina).
4
Nucleofection and Apoptosis Assay
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Amaxa® Cell Line Nucleofector® Kit V was purchased from Lonza Cologne AG (Germany). High glucose Dulbecco's modified Eagle's medium (DMEM) was from EuroClone S.p.A. (Italy). EPI was purchased from Selleckchem (USA). Anti-β-catenin antibody was purchased from Millipore (USA). A specific antibody against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was taken from NeoBioLab (USA). Anti-phosphorylated Akt (Ser473, Ser472 and Ser474) antibody was purchased from R&D System (USA) and anti-rabbit secondary horseradish peroxidase (HRP) antibody was purchased from Santa Cruz Biotechnology (USA). Annexin V-FITC apoptosis detection kit was from BioVision (USA). LDH-cytotoxicity Assay Kit was purchased from Abcam (UK).
5
Immunoprecipitation of LINC01278 RNA
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Cells were xed and crosslinked in 1% formaldehyde, and lysed using RIPA buffer supplemented with protease-inhibitor cocktail and RNase inhibitor. The cell lysates were incubated with magnetic beads conjugated with anti-β-catenin antibody (Millipore). Mouse IgG (Millipore) was used as the negative control. The immunoprecipitated RNA was extracted from the eluate, and the enrichment of LINC01278 or control ACTB was analyzed by quantitative RT-PCR.
6
Embryo Processing for Immunohistochemistry
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To control for technical variation, control and transgenic embryos were processed in parallel, using identical procedures and reagents in a 24-well plate. Probe details are available in supplementary material Table S3. For immunohistochemistry, embryos were fixed in 2% (β-catenin) or 4% (dpErk) paraformaldehyde in 1×PBS at 4°C, washed in PBST (PBS with 0.1% Tween 20) and dehydrated overnight in methanol at -20°C. After progressive rehydration in PBST they were deyolked (β-catenin) and blocked in 10% sheep serum, 10 mg/ml BSA in PBS with 0.5% Triton X-100 (β-catenin) or 0.1% Tween 20 (dpErk). Anti-β-catenin antibody (Sigma, clone 15B8) was diluted 1/300 and goat anti-mouse IgG-coupled Alexa594 (Molecular Probes) and Hoechst 34222 were diluted 1/1000. Mouse monoclonal anti-dpErk [which recognizes Erk1/2 (Mapk3/1) in their double-phosphorylated state; gift of Ben Shilo, Weizmann Institute, Israel] was diluted 1/2000.
7
Construction and Biochemical Validation of TBL1XR1 Mutant
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The FLAG-tagged TBL1XR1 expression construct was a gift from Dr. Cun-Yu Wang (University of California, Los Angeles)17 (link). The FLAG-tagged mutant TBL1XR1 (TBL1XR1Phe10Leu) expression construct was made by a PCR-based mutagenesis protocol43 (link). For biochemical experiments, the following antibodies were used: rabbit polyclonal anti-β-catenin antibody (Sigma, Beverly, MA, USA), rabbit polyclonal anti-N-CoR antibody (EMD Millipore, Billerica, MA, USA), as well as mouse monoclonal and rabbit polyclonal anti-FLAG antibodies (Sigma, Beverly, MA, USA).
8
Wnt/β-Catenin Signaling Pathway Modulation
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DMSO, anti‐β‐catenin antibody, RPMI 1640, and pyrvinium pamoate were from Sigma‐Aldrich (St. Louis, MO). Complete protease inhibitor cocktail (Complete) was from Roche Diagnostics (Tokyo, Japan). CHIR‐99021 was from Focus Biomolecules (Plymouth Meeting, PA). PPKF115‐584 was from BioVision, Inc (Milpitas, CA). Immobilon membrane was from Millipore (Bedford, MA). The Kras inhibitor SAH‐SOS1A was from Merck (Darmstadt, Germany). Anti‐Lamin B1, anti‐C/EBPα, anti‐C/EBPɛ, anti‐calcium pump pan PMCA ATPase (PMCA), anti‐G‐CSF receptor antibodies, and AKT inhibitor (AKTi‐1/2) were from Abcam (San Francisco, CA). Anti‐GSK3β, anti‐p‐GSK3β (Ser9), and anti‐Tcf4/Tcf7L2 antibodies were from Cell Signaling Technology (Danvers, MA). Anti‐G‐CSF antibody was from R&D Systems (Minneapolis, MN). Human Kras and control siRNAs were from Dharmacon (Lafayette, CO). PE‐labeled mouse IgG1k isotype control and PE‐labeled anti‐human CD11b were from eBioscience, Inc (San Diego, CA). Nuclear Extract Kits were from Active Motif (Carlsbad, CA), and Kras Activation Assay Kits were from Cell Biolabs, Inc (San Diego, CA). The luciferase assay system was from Promega Corporation (Madison, WI). Plasmids of Super8xTOPFlash (M50) and Super8xFOPFlash (M51) were kind gifts from Dr. Craig C. Malbon (State university of New York at Stony Brook, Stony Brook, NY).
9
Immunohistochemical Analysis of β-Catenin in Intestinal Tumors
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Immunohistochemistry were performed on 3 µm thick paraffin-embedded sections of the small intestinal tumors with anti-β-catenin antibody. The sections were deparaffinized in xylen and re-hydrated through graduated ethanol at room temperature. Tissue sections were soaked in 10 mM citrate buffer (pH6.0) and then subjected to antigen retrieval by microwaving for 20 minutes before the primary antibodey reaction. After treatment with 3% hydrogen peroxide for 5 minutes and 50 mM Tris-HCl (pH7.5) containing 1% BSA for 15 minutes, a 1:4000 diluted rabbit polyclonal anti-β-catenin antibody (Sigma) was applied to the sections, and incubated either at room temperature for 1 hr or at 4°C overnight. The detections were carried out with avidin-biotin-enzyme complex (ABC) method using LSAB+ System-HRP (DAKO) according to the manufacturer's protocol. The tumors with more than 5% of nuclear stained cells were counted for positive.
10
Zebrafish Embryo In Situ Hybridization
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Zebrafish embryos that reached the desired stages were fixed in 4% paraformaldehyde. Digoxigenin-labeled antisense RNA probes were used for in situ hybridization. In situ hybridization and immunofluorescence in zebrafish embryos were performed essentially as before (66 (link)). Anti-GFP antibody (1:1000 dilution; Santa Cruz Biotechnology, sc-9996) and anti–β-catenin antibody (1:1000 dilution; Sigma-Aldrich, C2206-1ML) were used for immunofluorescence. Embryos were observed by confocal microscopy with a ZEISS LSM710 META microscope.
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