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Recombinant human wnt3a protein

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Recombinant human Wnt3a protein is a secreted glycoprotein that belongs to the Wnt family of proteins. It is produced in a human cell expression system and purified. The protein functions as a signaling molecule involved in various biological processes.

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Lab products found in correlation

Imager 6000, by MSD (1 mentions) Lithium chloride (licl), by Merck Group (1 mentions) Xav939, by Selleck Chemicals (1 mentions) Epz 5676, by Chemietek (1 mentions) Dot1l sirna, by Thermo Fisher Scientific (1 mentions) Ex 527, by Merck Group (1 mentions) Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions) Hiperfect, by Qiagen (1 mentions) Dual luciferase reporter assay system, by Promega (1 mentions) Penicillin streptomycin solution 100, by Fujifilm (1 mentions) L glutamine, by Thermo Fisher Scientific (1 mentions) Enhanced chemiluminescence ecl substrate, by Thermo Fisher Scientific (1 mentions) Bicinchoninic acid bca assay kit, by Thermo Fisher Scientific (1 mentions) Streptavidin alexa fluor 555, by Thermo Fisher Scientific (1 mentions) α tubulin antibody, by Santa Cruz Biotechnology (1 mentions) β catenin, by Santa Cruz Biotechnology (1 mentions) Dimethyl sulfoxide (dmso), by Merck Group (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions) Blebbistatin, by Abcam (1 mentions) Bendamustine, by Selleck Chemicals (1 mentions)

14 protocols using recombinant human wnt3a protein

1

Quantitative WNT3A Protein Assay

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Cells were cultured on Matrigel in neural induction medium with or
without DOX. After 24 h, supernatant was collected and cells were scraped in
lysis buffer. WNT3A protein levels were assayed using a Mesoscale Discovery
system (MSD) WNT3A kit following the manufacturer’s protocol. Plates were
analyzed on a SECTOR Imager 6000 instrument with MSD Workbench™ v.3
software. Human WNT3A recombinant protein (R&D Systems) was used as
standard.
Jeon K., Kumar D., Conway A.E., Park K., Jothi R, & Jetten A.M. (2018). GLIS3 Transcriptionally Activates WNT Genes to Promote Differentiation of Human Embryonic Stem Cells into Posterior Neural Progenitors. Stem cells (Dayton, Ohio), 37(2), 202-215.
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2

Implantation of Wnt3a-coated Beads

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Intracutaneous implantation of protein-coated beads was performed as described (30 (link)). Human Wnt3a recombinant protein (R&D systems) was re-suspended in 1mg/ml BSA solution at 1mg/ml. Affinity affi-gel blue gel beads (Bio-Rad) were then suspended in 5µl protein solution, either control (1mg/ml BSA) or experimental (1mg/ml Wnt3a), at 4°C for 2hrs. Approximately 100 beads were implanted into skin immediately after irradiation. Mice were sacrificed at different time points post-irradiation. To visualize HFs, skin specimens were dehydrated in ethanol with graded concentrations and then immersed in xylene until it became transparent. The skin specimens were then further processed for histological examination and immunofluorescent staining.
Huang W.Y., Lai S.F., Chiu H.Y., Chang M., Plikus M.V., Chan C.C., Chen Y.T., Tsao P.N., Yang T.L., Lee H.S., Chi P, & Lin S.J. (2017). Mobilizing transit-amplifying cell-derived ectopic progenitors prevents hair loss from chemotherapy or radiation therapy. Cancer research, 77(22), 6083-6096.
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3

Inhibition and Knockdown of DOT1L in Cell Lines

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The specific DOT1L inhibitor EPZ-5676 was obtained from Chemietek. Lithium chloride and EX527 were purchased from Sigma, XAV-939 from Selleck and SRT1720 from Calbiochem. HiPerFect and all siRNAs except DOT1L siRNA were purchased from Qiagen (Supplementary Table 2). DOT1L siRNA and lipofectamine RNAimax were purchased from Invitrogen. Recombinant human WNT3A protein was purchased from R&D Systems.
Monteagudo S., Cornelis F.M., Aznar-Lopez C., Yibmantasiri P., Guns L.A., Carmeliet P., Cailotto F, & Lories R.J. (2017). DOT1L safeguards cartilage homeostasis and protects against osteoarthritis. Nature Communications, 8, 15889.
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4

Regulation of Wnt Signaling by BCL-3

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BCL-3 expression was suppressed via RNAi and TOPFlash assay was performed as previously described (Petherick et al., 2013 (link)) using Promega Dual-Luciferase Reporter Assay System (Promega, WI, USA) according to the manufacturer's instructions. Luminescence was measured at 560 nm using a Modulus luminometer (Turner Biosciences, CA, USA). Co-transfection of FOPFlash reporter with mutated TCF consensus sites was used alongside TOPFlash to monitor non-specific output. In RKO cells, 100 ng/ml recombinant human WNT3a protein (R&D Systems, Abingdon, UK) was added to cells 48 h post-siRNA transfection. For BCL-3 transient overexpression, cells were transfected with pcDNA3 control and pcDNA3-BCL-3 WT plasmids prior to Wnt3a treatment.
Legge D.N., Shephard A.P., Collard T.J., Greenhough A., Chambers A.C., Clarkson R.W., Paraskeva C, & Williams A.C. (2019). BCL-3 promotes a cancer stem cell phenotype by enhancing β-catenin signalling in colorectal tumour cells. Disease Models & Mechanisms, 12(3), dmm037697.
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5

Investigating CHIR99021 and DCA Effects on Colon Cancer Cell Lines

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HCT 116 cells were treated with CHIR99021 (Stemgent) at 3.3µM for 16 hours in serum-free medium. HT-29 cells were treated with CHIR99021 at 6.7µM for 24 hours in serum-free medium. And HCT 116 cells and HT-29 cells were treated with DCA (Sigma-Aldrich) at 10µM for 2hours in serum-free medium. HT-29 cells were treated with recombinant human Wnt3A protein (R&D system) at 300ng/ml for 24 hours after 24 hours serum starvation.
Huang S., Fantini D., Merrill B.J., Bagchi S., Guzman G, & Raychaudhuri P. (2017). DDB2 is a Novel Regulator of Wnt-Signaling in Colon Cancer. Cancer research, 77(23), 6562-6575.
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6

Endoderm Differentiation of hiPSCs

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hiPSCs were induced to differentiate into endoderm (phase I) using 2mM L-Glutamine, 200 mM Solution (25030–081: ThermoFisher Scientific, Tokyo, Japan), 100 μM 2-mercaptoethanol (M3148-25ML:Sigma-Aldrich, St. Louis, MO), 1% Penicillin-Streptomycin Solution (×100) (16823191: FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), 100 ng/mL Recombinant Human/Mouse/Rat Activin A Protein (338-AC-010: R&D Systems, Minneapolis, MN), 25 ng/mL Recombinant Human Wnt-3a Protein (5036-WN-010: R&D SYSTEMS, Minneapolis, MN), 0.29% Albumin, Human, recombinant expressed in plants (018–21541: FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) in RPMI 1640 with L-Gln, liquid medium (30264–85: Nacalai Tesque, Kyoto, Japan) for 24 h.
Nakashima Y., Miyagi-Shiohira C., Saitoh I., Watanabe M., Matsushita M., Tsukahara M, & Noguchi H. (2022). Induced hepatic stem cells are suitable for human hepatocyte production. iScience, 25(10), 105052.
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7

Wnt-3a Signaling Pathway Assay

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Dimethyl sulfoxide (DMSO) and 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) were purchased from Sigma‐Aldrich (St. Louis, MO, USA). Recombinant human Wnt‐3a protein was purchased from R&D systems (Minneapolis, MN, USA). β‐catenin, α‐tubulin antibodies, horseradish peroxidase‐conjugated mouse IgGκ binding protein (m‐IgGκ BP‐HRP), and CruzFluor 488‐conjugated mouse IgGκ binding protein (m‐IgGκ BP‐CFL 488) were obtained from Santa Cruz Biotechnology (CA, USA). The bicinchoninic acid (BCA) assay kit and enhanced chemiluminescence (ECL) substrate were obtained from Thermo scientific (Rockford, IL, USA). Alexa Fluor 555 streptavidin was obtained from Invitrogen (CA, USA), and 4,6′‐diamidino‐2‐phenylindole (DAPI) was purchased from Vector Laboratories (Burlingame, CA, USA).
Won A., Choi S., Kim A, & Hong J. (2023). Effect of DNA aptamer through blocking of negative regulation of Wnt/β‐catenin signaling in human hair follicle dermal papilla cells. Skin Research and Technology, 29(5), e13326.
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8

Wnt3a and PTGDS Protein Regulation

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Recombinant human Wnt3a protein was obtained from R&D Systems (5036-WN, MN, USA) and recombinant human PTGDS protein (rhPTGDS) was bought from GenWay Biotech (GWB-AC3EE7, CA, USA). Adriamycin, bendamustine, and WP1066 were purchased from Selleck Chemicals (TX, USA). AT56 was from Cayman (13160, MI, USA) and Blebbistatin was bought from Abcam (ab120425, MA, USA). Tunicamycin (ab120296, Abcam) and PNGase F (P0704S, New England Biolabs, MA, USA) were purchased, respectively.
Hu S., Ren S., Cai Y., Liu J., Han Y., Zhao Y., Yang J., Zhou X, & Wang X. (2021). Glycoprotein PTGDS promotes tumorigenesis of diffuse large B-cell lymphoma by MYH9-mediated regulation of Wnt–β-catenin–STAT3 signaling. Cell Death and Differentiation, 29(3), 642-656.
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9

Wnt-3a Cell Culture Protocol

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L-Wnt-3a-cells (ATCC® CRL-2647™) were acquired from ATCC. Cell culture reagents and their supplies: PNIPAAm-PEG polymers (Mebiol® Gel, Cosmo Bio, USA); Trypsin-EDTA 0.05% (Invitrogen); Trypsin inhibitor (SIGMA); LIVE/DEAD® Cell Viability staining (Invitrogen). Luciferase assay kit (Biovision, K801-200). DMEM (GE Healthcare Life Sciences); FBS (Atlanta biologicals); Recombinant human Wnt3A protein (R&D systems). Trypan blue solution was obtained from Sigma-Aldrich.
Li Q., Wang Q., Wang O., Shao K., Lin H, & Lei Y. (2018). A simple and scalable hydrogel-based system for culturing protein-producing cells. PLoS ONE, 13(1), e0190364.
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10

Cell Culture and Reagent Preparation

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The HEK293T (human embryonic kidney cell), CT26 (murine colon cancer), RENCA (murine renal cell carcinoma), and A20 (murine lymphoma) cell lines were purchased from ATCC (Manassas, VA). The HEK293T and CT26 cell lines were maintained in DMEM (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) supplemented with 10% foetal calf serum (FCS; Cytiva, Tokyo, Japan). The RENCA and A20 cell lines were maintained in RPMI medium (FUJIFILM Wako Pure Chemical Corporation) supplemented with 10% FCS. All tumour cells were confirmed to be Mycoplasma (−) using a PCR Mycoplasma Detection Kit (TaKaRa, Shiga, Japan) according to the manufacturer’s instructions before use. Recombinant human Wnt3a protein was obtained from R&D (Minneapolis, MN). Recombinant murine Wnt3a protein was purchased from PeproTech (Cranbury, NJ). Rat anti-mouse PD-1 mAb (RMP1-14) and control rat IgG2a mAb (RTK2758) were obtained from BioLegend (San Diego, CA).
Ishino T., Kawashima S., Tanji E., Ueno T., Ueda Y., Ogasawara S., Sato K., Mano H., Ishihara S., Kato N., Kawazu M, & Togashi Y. (2023). Somatic mutations can induce a noninflamed tumour microenvironment via their original gene functions, despite deriving neoantigens. British Journal of Cancer, 128(6), 1166-1175.
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