Cells were seeded into 8-chamber culture slides and grown for 24 h fixed with 100% methanol and 100% acetone at −20 °C. The cells were subsequently treated with β-catenin antibodies (Cell Signaling Technology, Danvers, MA, USA) and incubated with Alexa Flour 488 conjugate secondary antibodies. Mounting medium containing DAPI was then added. Confocal fluorescence images were acquired with a laser scanning microscope (LSM 700; Zeiss, New York, NY, USA).
β catenin antibody
Manufactured by Cell Signaling Technology
Sourced in United States, China
β-catenin antibody is a laboratory tool used for the detection and analysis of β-catenin, a key signaling molecule involved in various cellular processes. This antibody can be used in techniques such as Western blotting, immunohistochemistry, and immunofluorescence to identify and quantify the presence of β-catenin in biological samples.
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Lab products found in correlation
Lsm 780 confocal microscope, by Zeiss (6 mentions)
E cadherin antibody, by Cell Signaling Technology (5 mentions)
Alexa fluor secondary antibody, by Thermo Fisher Scientific (4 mentions)
Snail slug antibody, by Abcam (4 mentions)
N cadherin antibody, by Abcam (4 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (3 mentions)
Lsm 700, by Zeiss (2 mentions)
Signalstain dab substrate kit, by Cell Signaling Technology (2 mentions)
Anti ki67 antibody, by Abcam (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Oct 4 antibody, by Cell Signaling Technology (2 mentions)
Sox2 antibody, by R&D Systems (2 mentions)
Nanog antibody, by Bioss Antibodies (2 mentions)
A1r inverted laser scanning confocal microscope, by Nikon (2 mentions)
Facscalibur, by BD (2 mentions)
Cellquest pro software, by BD (2 mentions)
Alexa fluor 594 goat anti mouse igg h l, by Thermo Fisher Scientific (2 mentions)
Paraformaldehyde solution, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 goat anti rabbit igg h l, by Thermo Fisher Scientific (2 mentions)
Htert antibody, by Abcam (2 mentions)
50 protocols using β catenin antibody
1
Immunofluorescent Staining of β-Catenin
2
Breast Cancer Cell Line Maintenance
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Human breast cancer cells MCF7, MDA-MB-231, T47D, MDA-MB-453, and human mammary epithelial cell line MCF10A were obtained from American Type Culture Collection. All breast cancer cells were maintained in high glucose DMEM (Invitrogen) supplemented with 10% fetal bovine serum, 100 units/mL penicillin and 100 μg/mL streptomycin. MCF-10A was cultured in DMEM/Ham’s F-12 medium supplemented with growth factors and 100 ng/ml Cholera Toxin. All cells were incubated in a 5% CO2 at 37 °C. β-catenin antibodies were from Cell signaling technology (CST, Beverly, MA, USA); β-actin antibody were from sigma (St. Louis, MO, USA); GSK3β and PKM2 antibodies were from Signalway Antibody (SAB, College Park, MD, USA); laminB antibodies were from Epitomics (Burlingame, CA, USA). The IGF-1 cytokine was purchased from sigma.
3
Immunohistochemical Analysis of JAM3 and β-Catenin
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Immunohistochemistry (IHC) was performed in primary EC samples and paired adjacent tissue samples. JAM3 antibody (Abcam, USA), phosphorylated β-catenin (p-β-catenin) antibody (ZENBIO, China) and β-catenin antibody (Cell Signaling Technology, USA) were diluted to 1:25, 1:30 and 1:500, respectively. For antigen retrieval, the slides were placed in citrate antigen-repairing solution and heated in a high-pressure cooker until steam arose. The slides were kept inside the cooker for 150 s and then cooled down at room temperature for 15 min. The intensity and the scope of staining were scored using the German semiquantitative scoring system as described previously [40 (link), 41 (link)].
4
Ubiquitination and Turnover of β-Catenin
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For ubiquitination assays, HA-ubiquitin plasmids were transfected into 293FT cells. After pretreat with 100 μM DHC or DMSO for 16 h, 20 µg/ml proteasome inhibitor MG132 (Sigma-Aldrich) was added to the cells and incubated for another 8 h. Then the cells were collected and lysed and equal amount of protein from each sample was incubated with β-catenin antibody (1:100; Cell Signaling) overnight at 4°C. Protein A/G Magnetic Beads (Beyotime) were added to adsorb the immunoprecipitants and suspended with 1× loading buffer for western blotting. Ubiquitin antibody (1:1,000; Abcam) was used to check the interaction between β-catenin and ubiquitin. For the turnover assay, DHC and DMSO pretreated A375 cells were incubated with 50 µg/ml cycloheximide (CHX, Sigma-Aldrich) for indicated time in the presence or absence of DHC. Then, cells were collected, lysed and the protein levels of β-catenin were assessed by western blotting.
5
β-Catenin Translocation in T Cells
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To identify the translocation of β-catenin from the cytoplasm to the nucleus after T cells were treated with TWS119, cells were fixed in methanol, blocked using 1% bovine serum albumin (BSA) dissolved in 0.1% Triton X-100, and incubated with rabbit monoclonal β-catenin antibody [1:100; Cell Signaling Technology, 8480(CST)] at 4°C overnight. An anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 594 Conjugate, 1:1000; CST, 19158) was used to visualize the binding sites of the primary antibody. Cells were observed by immunofluorescence microscopy using a DMIRE2 microscope (Leica). 4′, 6-Diamidino-2-phenylindole (DAPI) Fluoromount-G (Southern Biotech) was used to stain cell nuclei.
6
Westerns Blot Analysis of Murine Epididymal Proteins
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For Western blots, murine epididymal proteins were pooled from different developmental ages (six animals per age group) and the other organ proteins were pooled from five adult mice. Total proteins were extracted, and Western blot analysis was performed as described previously.17 (link) Immuno-blots were developed with ECL reagent (Pierce Biosciences, Rockford, IL, USA) according to the manufacturer's instructions. There were no nonspecific bands on the gels when incubated with antibodies against β-catenin and Gapdh (data not shown).
For quantification, densitometric analysis was performed on the gray level intensity of target bands derived from scanned films, processed by using Gene Tools image analysis software (GeneTools, version 4.02; Syngene, Cambridge, UK) according to Wang et al.17 (link) The primary antibodies used were β-catenin antibody (9587, Cell Signalling Technology, Danvers, MA, USA) and Gapdh antibody (2118, Cell Signalling Technology).
For quantification, densitometric analysis was performed on the gray level intensity of target bands derived from scanned films, processed by using Gene Tools image analysis software (GeneTools, version 4.02; Syngene, Cambridge, UK) according to Wang et al.17 (link) The primary antibodies used were β-catenin antibody (9587, Cell Signalling Technology, Danvers, MA, USA) and Gapdh antibody (2118, Cell Signalling Technology).
7
Dissecting cGAS-STING Signaling Pathway
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dUTP was from Sigma, RNA polymerase III inhibitor (ML-60218) was from Merck Millipore, dNTP (Fermentas), type I IFNα were from PBL Biomedical Laboratories, CpG (Invitrogen), polyIC and peptidoglycan (from Staphylococcus aureus, InvivoGen), Taq Polymerase (BIOTAQ DNA polymerase, Bioline), Pfu (Agilent Technologies), cGAS siRNA (sc-95512), pol III RPC39 siRNA (sc-36292), pol III RPC62 siRNA (sc-76188) were from Santa Cruz Biotechnology, STING (antibody #3337), phospho-IRF-3 (Ser396) (4D4G, rabbit mAb #4947), IRF-3 (D6I4C, XP rabbit mAb #11904), β-catenin antibody (amino-terminal antigen, #9581), mouse anti-rabbit IgG (conformation specific, L27A9, mAb #3678), anti-rabbit IgG, HRP-linked (antibody #7074), cGAS (D1D3G, mAb#15102) antibodies were from Cell Signaling Technology. APOBEC3A antibody (SAB4500753) was from Sigma Aldrich. RIG-I (E-5, sc-376882), pol III RPC39 (C 39-2, sc-23913), pol III RPC62 (I-18, sc-69534) antibodies were from Santa Cruz Biotechnology, Inc. Anti-mouse IgG, (HRP-linked antibody #NA931V) was from GE Healthcare. Monoclonal anti-β-actin−peroxidase (antibody #A3854) was from Sigma Aldrich. Human IFNα (IgA, mba-hifnα-3), hIFNβ (IgG, mbg-hifnβ-3), hIFNγ (IgA, mba-hifnγ-3) and Mouse IgG2a (mbg2a-ctrlm) antibodies used as control were from InVivogen.
8
Epithelial-Mesenchymal Transition Protein Analysis
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Western blot was performed to analyze the expression of proteins. The following antibodies were used for this analysis, E-cadherin antibody, Vimentin antibody, β-catenin antibody, Snai antibody, Slug antibody, Twist antibody, (Cell Signaling Technology, Beverly, MA). Protein expression was quantifed by densitometric analysis, and the expression levels were normalization against that of β-actin (Sigma Aldrich).
9
Immunological Profiling of Virus-Expressing Cells
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Wild type and mutant viruses expressing HEK-293T cells were cultured in DMEM with 5% bovine growth serum (Gibco, Carlsbad, CA). De-identified PBMCs were provided from the Human Immunology Core at the University of Pennsylvania. The core maintains an IRB approved protocol in which the declaration of Helsinki protocols was followed and each donor gave written informed consent. PBMCs were maintained in RPMI with 10% fetal bovine serum (FBS) (Hyclone, South Logan, Utah). A10 (EBNA3C), and S12 (LMP1) hybridomas were described previously [32 (link), 33 (link)]. BZLF1 antibody was provided by Martin Rowe (University of Birmingham, UK) [34 (link)]. EBNA1 antibody was purchased from Advanced Biotechnologies, Inc., Columbia, MD. β-Catenin antibody was procured from Cell Signaling, Inc. Beverly, MA. Rabbit polyclonal RBP-Jk antibody was provided by Elliott Kieff (Harvard Medical School, Boston, MA). Nm23-H1 antibody was procured from Seikagaku Corp (Tokyo, Japan). The p-ERK (E-4), p-JNK (G-7), p-P38 (D-8) antibodies were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA), and the GAPDH antibody was obtained from US-Biological Corp. (Swampscott, MA).
10
Quantitative Analysis of β-Catenin Signaling
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HUVECs from each group were added in RIPA lysis buffer (Beyotime, P0013B, China). Protein concentrations were measured using a BCA Protein Assay kit (KeyGEN Biotech, China). Proteins were separated on 10% SDS-polyacrylamide gels assayed by immunoblot using β-catenin antibody (Cell Signaling Technology, 8480S, USA), p-β-catenin (Ser675) antibody (Cell Signaling Technology, 9567S, USA), GSK3β (Ser9) antibody duet (Cell Signaling Technology, 8213S, USA), and β-actin antibodies (ZSGB-Bio, PR-0255, China). Secondary antibody used was (HRP)-conjugated goat anti-rabbit or anti-mouse IgG (ZSGB-Bio, ZB-2305, ZB-2301, China) at 1:5000. Signals were visualized with super enhanced chemiluminescence (ECL) detection reagent (BOSTER, AR1172, China) and detected by Quantity One analysis system (Bio-Rad).
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