β catenin

Manufactured by Santa Cruz Biotechnology

Sourced in United States, United Kingdom, China, Germany, Morocco, Australia

β-catenin is a protein that plays a central role in the Wnt signaling pathway. It functions as a key mediator in the canonical Wnt pathway, which regulates various cellular processes such as cell-cell adhesion and gene transcription. β-catenin is involved in the regulation of cell-cell adhesion and acts as an intracellular signal transducer in the Wnt signaling pathway.

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Lab products found in correlation

413 protocols using β catenin

Peptide Enhances Hair Growth

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Example 2

Human hair follicle dermal papilla cells were seeded at a density of 4×10⁵cells/well on 6-well plates, followed by incubation overnight. After changing into serum-free medium, the cells were treated with the peptides, followed by incubation for 15 and 30 minutes, and then the wells were harvested to isolate nuclear and cytoplasmic proteins. Western blotting was performed using β-catenin (Santa Cruz Biotechnology, USA) to compare β-catenin expression patterns. The results are shown in FIGS. 2a and 2b.

As can be confirmed from FIGS. 2a and 2b, the activity of β-catenin, which is a hair growth-related factor, was increased in human hair follicle dermal papilla by the treatment with the peptide composed of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. The increase in expression of β-catenin and the activity thereof increase the expression of hair growth-related molecules.

US10508140B2. Peptide having hair growth-promoting activity and/or melanin generation-promoting activity, and use thereof (2019-12-17). CAREGEN CO., LTD. [KR]. Inventors: Yong Ji Chung [KR], Eun Mi Kim [KR], Eung-Ji Lee [KR].

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Peptide Regulation of Hair Follicle Cells

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Example 2

As can be confirmed from FIGS. 2a and 2b, the activity of β-catenin and the phosphorylation level of GSK3β were increased in human hair follicle dermal papilla by the treatment with the peptide composed of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. The activity of β-catenin results from being free from a complex due to the degradation by GSKβ phosphorylation, and increases the expression of hair growth-related molecules.

US10344061B2. Peptide exhibiting hair growth promoting activity and/or melanin production promoting activity and use thereof (2019-07-09). CAREGEN CO., LTD. [KR]. Inventors: Yong Ji Chung [KR], Eun Mi Kim [KR], Eung Ji Lee [KR].

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Immunofluorescent Staining of Adherent Cells

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To stain AMs grown in culture, cells were grown on coverslips and fixed in fixation buffer (4% paraformaldehyde/2% sucrose/PBS) for 20 min at room temperature as previously reported (31 (link)). Coverslips were then rinsed twice in PBS and permeabilized in permeabilization buffer (0.4% Triton-X and 1% BSA in PBS) for 20 min. Primary antibodies B-Catenin (Santa Cruz) and Stat3 (Cell Signaling) were applied at 1:250 dilution in staining buffer (0.1% Triton-X and 0.1% BSA in PBS) overnight at 4°C in a humid chamber. Coverslips were washed 5 times (5 min each) in wash buffer (0.2% Triton-X and 0.2% BSA in PBS). Secondary antibodies (AlexaFluor secondary 488 or 594, Invitrogen) were applied at 1:250 dilution in staining buffer for 2–3 hours at room temperature in a humid chamber in the dark. Prior to mounting with Vectorshield with DAPI (Vector Laboratories, CA), coverslips were washed two more times in wash buffer. Immunofluorescent analysis was conducted on an Olympus TH4–100 fluorescent microscope using Slidebook V.4.1 digital microscopy.

Scortegagna M., Du Y., Bradley L.M., Wang K., Molinolo A., Ruppin E., Murad R, & Ronai Z.A. (2023). Ubiquitin ligases Siah1a/2 control alveolar macrophage functions to limit carcinogen-induced lung adenocarcinoma. Cancer research, 83(12), 2016-2033.

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Immunohistochemical Analysis of EMT Markers

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Formalin fixed paraffin tissue sections (4 μm) were used with an indirect peroxidase labeling technique (Envision Plus, DAKO, Australia). Following deparaffinization and rehydration, endogenous peroxidase activity was blocked with 3 % H₂O₂ and non-specific binding inhibited with 10 % normal goat serum (01–6201 Zymed Laboratories, USA). Heat induced epitope retrieval was used. Antigens were visualised by immunohistochemical staining using their respective antibodies diluted as follows: (CD34 1:500, AbD serotec MCA18256; Ki-67 1:100, Thermoscientific, RM-9106-S1; caspase 3 1:800, R&D AF835) and EMT markers (ZEB1 1:200, Santa Cruz sc-25388; Vimentin 1:300, Santa Cruz sc-5565; E-cadherin 1:500 Sana Cruz sc-7870; b-catenin 1:300, Santa Cruz sc-7199) Negative controls were stained by the corresponding isotype antibodies. Following primary antibody treatment, sections were incubated with a horseradish peroxidase labelled polymer secondary antibody. The antigen-antibody complex was visualized by diaminobenzene (DAB) Peroxidase Substrate Solution (DAKO, Australia). Each treatment group consisted of 5–8 animals. A minimum of ten tumors were assessed for each treatment group and from 3 to 15 images per tumor, depending on tumor size, were captured for analysis.

Nguyen L., Fifis T, & Christophi C. (2016). Vascular disruptive agent OXi4503 and anti-angiogenic agent Sunitinib combination treatment prolong survival of mice with CRC liver metastasis. BMC Cancer, 16, 533.

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Investigating Oxidative Stress and EMT Regulation

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All culture materials were purchased from Gibco (Grand Island, NY, USA). 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), ROS scavenger (N-acetyl cysteine [NAC]), 2, 7-dichlorodihydrofluorescein diacetate (H2DCFDA), dihydroethidium (DHE), phosphoinositide 3-kinase inhibitor (wortmannin) and p38 MAPK inhibitor (SB203580) were purchased from Sigma (St. Louis, MO, USA). Mouse monoclonal antibodies against PRDX6, N-cadherin, b-catenin, Vimentin, Slug, Snail, Twist-1 and acetylation of H3 (Ac-Histone H3) at Lys 9 and Lys 14, and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit polyclonal antibodies against ERK1/2Thr 202 Ty r204 , p38 Thr 180 Tyr 182 , and phosphoinositide 3-kinase Tyr 458 mouse/rabbit monoclonal antibody were purchased from Cell Signaling Technology (Beverly, MA, USA). The TdT-mediated dUTP Nick End Labeling (TUNEL) kits were from Roche (Germany). SDS, NP-40, sodium deoxycholate, protease inhibitor cocktails were purchased from Sigma (St. Louis, MO, USA). SDS, NP-40, sodium deoxycholate, protease inhibitor cocktails were purchased from Sigma.

Huang W.S., Huang C.Y., Hsieh M.C., Kuo Y.H., Tung S.Y., Shen C.H., Hsieh Y.Y., Teng C.C., Lee K.C., Lee K.F, & Kuo H.C. (2018). Expression of PRDX6 Correlates with Migration and Invasiveness of Colorectal Cancer Cells. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology, 51(6).

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ChIP-qRT-PCR for Protein-DNA Interactions

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Chromatin immunoprecipitation (ChIP) assays were carried out as previously described with minor modifications [25] . Briefly, harvested EBs were washed with PBS and incubated in 1% formaldehyde for 15 minutes at RT. Then, they were quenched by the addition of 125 mM glycine for 5 minutes at RT. Crosslinked cells were lysed in SDS lysis buffer, and DNA was sonicated. Chromatin was subjected to immunoprecipitation with a mixture of protein A-and G-agarose (GE Healthcare Life Sciences, UK, www.gelifesciences.com) at 4 C. Each primary antibody was incubated overnight with chromatin at 4 C. Antibodies used for the ChIP assay were as follows; SMAD1 (Cell Signaling Technologies; #9743), and b-CATENIN (Santa Cruz Biotech; SC31000). The chromatin-antibody-agarose complexes were then sequentially washed with the following solutions; low salt wash buffer, high salt wash buffer, LiCl buffer, and TE buffer. The chromatin complexes were eluted, 300 mM NaCl was added to the eluates, and reverse histone-DNA cross-linking was performed by heating at 68 C. Finally, the DNA/chromatin solution was treated with proteinase K and RNase A, and the DNA was isolated using phenol/chloroform extraction, precipitated with ethanol, and resuspended in 50 ml DW. The resulting DNA was used for qRT-PCR analyses.

Han K.M., Kim S.K., Kim D., Choi J.Y., Im I., Hwang K.S., Kim C.H., Lee B.H., Yoo H.W, & Han Y.M. (2015). Enhanced SMAD1 Signaling Contributes to Impairments of Early Development in CFC-iPSCs. Stem cells (Dayton, Ohio), 33(5).

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Western Blot Analysis of Cell Signaling Proteins

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Western blotting procedure was performed as described previously [11] . Briefly, eight samples of protein extracts from each group (50 mg of protein per lane) were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis and blotted onto nitrocellulose membranes (BioRad, Hercules, CA, USA). We used Ponceau Red (Sigma, St. Louis, MO, USA) staining to control equal loading of gels. Primary antibodies against mouse Cx43 (CellSignaling, #3512), b-catenin (SantaCruz Biotechnology, #sc-1496), N-cadherin (Dako, #M3613), and a-tubulin (SantaCruz Biotechnology, #sc-5286) were used. Secondary antibodies were conjugated with horseradish peroxidase (anti-rabbit Star54, Serotec, Oxford, UK or anti-mouse, Sigma #A9309 as appropriate). Blots were visualized using enhanced chemiluminescence reaction (Pierce, Rockford, IL, USA), and exposed on the X-ray film (X-Omat Blue, Kodak, Rochester, NY). Then blots were incubated with the Restore 1 Western Blot Stripping Buffer (Thermo Scientific, Waltham, MA, USA). Films were scanned and quantified using the Image J software (National Institutes of Health, Bethesda, MD, USA). The abundance of Cx43, N-cadherin, and b-catetnin proteins were normalized to the level of a-tubulin. The results of particular experiments were related to the expression of proteins in the control group, which was set as 1.0.

Bonda T.A., Szynaka B., Sokołowska M., Dziemidowicz M., Winnicka M.M., Chyczewski L, & Kamiński K.A. (2016). Remodeling of the intercalated disc related to aging in the mouse heart. Journal of cardiology, 68(3).

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Protein Extraction and Western Blot Analysis

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The protein samples were extracted from osteoblasts via procedures that have previously been described in detail (Yang et al. 2007) (link). Protein samples (approximately 50 mg) were fractionated by SDS-PAGE (7.5-10% polyacrylamide gels). Separated proteins were blot transferred onto a nitrocellulose membrane. After blocking with 0.1% Tween 20 and 5% nonfat dry milk in Tris-buffered saline at room temperature for 1 h, the membrane was incubated overnight at 4 8C in one of the following primary antibodies as an internal control: FZD4 (1:400; Peprotech, Rocky Hill, NJ, USA), WNT2 (1:400; Santa Cruz Biotechnology, Santa Cruz, CA, USA), b-catenin (1:200; Santa Cruz Biotechnology), Runx2 (1:200; Santa Cruz Biotechnology), Osterix (1:400; Santa Cruz Biotechnology), and b-actin (1:1000; Santa Cruz Biotechnology). The membrane was incubated with HRP-conjugated secondary antibody (1:2000) for 1 h and detected using the ECL Western Blot System (Amersham Biosciences).

Shi C., Huang P., Kang H., Hu B., Qi J., Jiang M., Zhou H., Guo L, & Deng L. (2015). Glucocorticoid inhibits cell proliferation in differentiating osteoblasts by microRNA-199a targeting of WNT signaling. Journal of molecular endocrinology, 54(3).

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Protein Expression Analysis Protocol

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Cell lysates were prepared using RIPA buffer and analysed as described in [14 (link)]. Primary antibodies used were: MITF (Thermo Fisher Scientific), phospho-ERK (MAPK-YT) (SIGMA), ATF4 (NEB), CREB and phospho-CREB (Cell Signalling), ERK2, BRN2, beta-catenin, PAX3 and HIF1alpha (Santa Cruz Biotechnologies).

Ferguson J., Smith M., Zudaire I., Wellbrock C, & Arozarena I. (2017). Glucose availability controls ATF4-mediated MITF suppression to drive melanoma cell growth. Oncotarget, 8(20), 32946-32959.

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Comprehensive Protein Analysis Protocol

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Cells were prepared in lysis buffer containing 50 mM HEPES (pH 7.2), 150 mM NaCl, 25 mM beta-glycerophosphate, 25 mM NaF, 5 mM EGTA, 1 mM EDTA, 1% NP-40, 1 mM sodium orthovanadate, 0.1 mM PMSF, and a Protease Inhibitor cocktail (leupeptin, pepstatin, aprotinin, and antipain; each 5 μg/ml). For secretory protein preparation, the culture medium was centrifuged, and cellular components and debris were discarded. The culture medium was concentrated using 10 K cut-off microcon (Amicon), or by adding ice-cold acetone, the precipitated protein was resuspended in lysis buffer. The proteins were separated on SDS-PAGE and transferred to a 0.45-μm Immobilon P-transfer membrane (Millipore). The membrane was blocked in 5% (w/v) non-fat milk and then probed with a primary antibody; anti-human GLRX3 antibody, beta-catenin, E-cadherin, GAPDH (Santacruz, Dallas, Texas, US), c-MET, PI3K, pAKT (Cell signaling, Danvers, Massachusetts, US), AKT, Wnt1, 3, 5a, 7b,11, 16, RhoA, RhoB, pJNK, RAC1, Dvl2 (Santacruz, Dallas, Texas, US), and ABCG2 (Abcam, Cambridge, UK). The immunoreactive material was then visualized using SuperSignal West Pico Chemiluminescent substrate (Pierce Biotechnology, Rockford, Illinois, US) according to the manufacturer’s instructions.

Jo J.H., Kim S.A., Lee J.H., Park Y.R., Kim C., Park S.B., Jung D.E., Lee H.S., Chung M.J, & Song S.Y. (2021). GLRX3, a novel cancer stem cell-related secretory biomarker of pancreatic ductal adenocarcinoma. BMC Cancer, 21, 1241.

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