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Ab81484

Manufactured by Abcam
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Sourced in United Kingdom, United States

Ab81484 is a laboratory equipment product offered by Abcam. It is designed for a core function, but a detailed description cannot be provided while maintaining an unbiased and factual approach. Further information is not available.

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Lab products found in correlation

Rapamycin tlrl rap, by InvivoGen (1 mentions) Celltiter glo luminescent cell viability assay, by Promega (1 mentions) Celltiter 96 aqueous one solution cell proliferation assay, by Promega (1 mentions) Oleate, by Merck Group (1 mentions) Dual luciferase reporter dlr assay system, by Promega (1 mentions) Protein lysis buffer, by Promega (1 mentions) A939572, by Merck Group (1 mentions) Glomax 20 20 luminometer, by Promega (1 mentions)

5 protocols using ab81484

1

Investigating Wnt Pathway Modulation

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AllStars negative control siRNA and Stealth RNAi for NR3C1 were from Qiagen. CellTiter-Glo® Luminescent Cell Viability Assay (G7570) and CellTiter 96® AQueous One Solution Cell Proliferation Assay (G3582) were purchased from Promega. Matrigel® Basement Membrane Matrix, Growth Factor Reduced (GFR) was purchased from Corning. Antibodies against LRP5 (5731s), LRP6 (3395s), p-β-catenin (4176s), Cyclin D1 (2978s), LC3A/B (4108s) and p62 (5114 s) were purchased from Cell Signaling Technology. Antibodies against α-smooth muscle Actin (α-SMA) (ab5694), CD68 (ab53444), GAPDH (ab8245) and recombinant mouse Wnt3a protein (ab81484) were purchased from Abcam. Antibodies against CD31 (553370) and CD102 (553326) were purchased from BD Bioscience. Anti-GR (sc-393232) was purchased from Santa Cruz Biotechnology. Recombinant mouse sFRP3 protein (592-FR/CF) was purchased from Bio-techne. Rapamycin (tlrl-rap) was purchased from InvivoGen. Premo™ Autophagy Tandem Sensor RFP-GFP-LC3B Kit including chloroquine (P36239) was purchased from Thermo Fisher Scientific.
Liu B., Zhou H., Zhang T., Gao X., Tao B., Xing H., Zhuang Z., Dardik A., Kyriakides T.R, & Goodwin J.E. (2021). Loss of endothelial glucocorticoid receptor promotes angiogenesis via upregulation of Wnt/β-catenin pathway. Angiogenesis, 24(3), 631-645.
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2

Wnt3a Production and Application

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Wnt3a was produced from murine L cells. Cells were grown in a 10‐cm plate in DMEM supplemented with 10% FBS. When 90% confluence was reached, cells were diluted 1 : 10 and seeded in new 10‐cm plates. Wnt3a‐conditioned media were collected at 2, 4, and 6 days, aliquoted and stored at −20 °C until use. Recombinant Wnt3a was obtained from Abcam (ab81484, Cambridge, UK), dissolved in PBS supplemented with 0.25% BSA with 0.1 mg·mL−1 stock concentration and aliquoted for single use to avoid freeze/thaw cycles. Recombinant Wnt3a was added to the full growth media with 1 : 500 dilution (final concentration of 200 ng·mL−1). Cells were incubated in the Wnt3a‐enriched media for 6 h at 37 °C.
Sezgin E., Azbazdar Y., Ng X.W., Teh C., Simons K., Weidinger G., Wohland T., Eggeling C, & Ozhan G. (2017). Binding of canonical Wnt ligands to their receptor complexes occurs in ordered plasma membrane environments. The Febs Journal, 284(15), 2513-2526.
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3

Inhibition of Wnt Signaling Pathway

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Transfection cocktail was made using TransIT-Keratinocyte reagents according to the manufacturer's instruction (Mirus, WI, USA). After 16 hours, media was refreshed with inhibitors, A939572: 1 µM, TMP-153 sc-200649 : 0.5 µM, Oleate: 12.5 µM (Sigma). After 16 hours of inhibitor pretreatment, cells were refreshed with media containing inhibitors with or without Wnt3a (50 ng/mL) (ab81484, Abcam, MA, USA). After 24 hours, we harvested cells with 50 µL protein lysis buffer (Promega, WI, USA), freeze-thawed 3 cycles, and determined TOP Flash activity by dual-luciferase™ reporter (DLR™) assay systems (Promega) using spectrophotometer (Glomax 20/20 Luminometer, Promega).
Jain P., Nattakom M., Holowka D., Wang D.H., Brenna J.T., Ku A.T., Nguyen H., Ibrahim S, & Tumbar T. (2018). Runx1 role in epithelial and cancer cell proliferation implicates lipid metabolism and Scd1 and Soat1 activity. Stem cells (Dayton, Ohio), 36(10), 1603-1616.
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4

Asporin-mediated Wnt Signaling Modulation

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To supplement the Asporin protein in AVICs in culture, the cells were treated directly with the recombinant human Asporin protein (150 ng/ml; Abcam 132382) for 24 hrs in the presence of OS induction media for 4 days. For canonical Wnt signaling pathway manipulation, cultured cells were treated with the recombinant human Wnt3A (rhWnt3A) protein (150 ng/ml; Abcam ab81484) for 24 hrs to activate Wnt signaling, performing activation assay. Similarly, cultured cells were treated with Xav939, also for 24 hrs (2 μM; Abcam ab120897) for Wnt signaling inhibition assay [19 (link), 21 (link)]. After completion of all the treatments, experiments were carried out for either total RNA isolation for quantitative mRNA expression analyses or cell fixation for immunostaining. Also, qualitative and quantitative Alizarin Red staining was carried out to compare mineralization of treated cells with untreated controls (n = 3).
Polley A., Khanam R., Sengupta A, & Chakraborty S. (2020). Asporin Reduces Adult Aortic Valve Interstitial Cell Mineralization Induced by Osteogenic Media and Wnt Signaling Manipulation In Vitro. International Journal of Cell Biology, 2020, 2045969.
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5

Epithelial-Mesenchymal Transition Induction

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NMuMG cells and MDCK cells were cultured and grown to confluency as described above. To induce EMT, cells were starved in media containing 0.5% FBS and pretreated with pharmacological inhibitors 24 h prior to treatment with TGFβ1 at stated concentrations (R&D Systems, 240-B-010). Culture media was replaced every 24 h during all experiments. For some experiments, recombinant mouse Wnt3a (Abcam, ab81484) was used to stimulate serum starved NMuMG cells as above. For reversal, NMuMG cells were starved for 24 h, stimulated with TGFβ1 for 72 h, and then incubated with MDL-28170 at 10 or 20 µM in starvation media for an additional 72 h.
Kim D.H., Beckett J.D., Nagpal V., Seman-Senderos M.A., Gould R.A., Creamer T.J., MacFarlane E.G., Chen Y., Bedja D., Butcher J.T., Mitzner W., Rouf R., Hata S., Warren D.S, & Dietz H.C. (2019). Calpain 9 as a therapeutic target in TGFβ-induced mesenchymal transition and fibrosis. Science translational medicine, 11(501), eaau2814.
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