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Notch3

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Notch3 is a cell surface receptor that plays a critical role in cell-cell communication and signaling. It is a member of the Notch family of proteins, which are involved in regulating various cellular processes, including cell fate determination, proliferation, and differentiation. Notch3 functions as a transmembrane receptor, transmitting signals from the extracellular environment to the cell's interior.

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Lab products found in correlation

Vinculin, by Merck Group (2 mentions) Notch2, by Cell Signaling Technology (2 mentions) β actin, by Santa Cruz Biotechnology (2 mentions) β catenin, by Cell Signaling Technology (2 mentions) Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (2 mentions) Bca protein assay, by Thermo Fisher Scientific (2 mentions) Minigel, by Bio-Rad (1 mentions) Plexind1, by R&D Systems (1 mentions) E cadherin, by BD (1 mentions) Keap1, by Abcam (1 mentions) Jagged 2, by Cell Signaling Technology (1 mentions) Notch4, by Cell Signaling Technology (1 mentions) Jagged 1, by Cell Signaling Technology (1 mentions) Hif 1α, by Cell Signaling Technology (1 mentions) N cadherin, by Cell Signaling Technology (1 mentions) E cadherin, by Cell Signaling Technology (1 mentions) Snail1, by Cell Signaling Technology (1 mentions) Dual luciferase reporter assay kit, by Promega (1 mentions) Runx1, by Abcam (1 mentions) Runx1, by Cell Signaling Technology (1 mentions)

Spelling variants of this product

Anti-Notch3 (14 citations) Anti-NOTCH3 antibody (3 citations) Rabbit anti-NOTCH3 (2 citations) Notch3 primary antibody (2 citations) Rabbit monoclonal anti-Notch3 antibody (2 citations) Antibodies directed against Notch3 (2 citations) NOTCH3 (D11B8) (2 citations) Anti-NOTCH3 8G5 (2 citations)

12 protocols using notch3

1

Western Blot Analysis of Stem Cell Markers

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Western blot analysis was performed following standard procedures. Equal amount of protein was separated on 7.5% SDS–PAGE and transferred onto PVDF membrane. Western signals were detected using SuperSignal chemiluminescent substrate (West-Pico or West Dura Kits, Thermo Scientific). All of the uncropped western blots with molecular weight indicated were presented in Supplementary Figs. 9–14. Following antibodies were used in this study: β-catenin (1:1000, 8480 and 9562) phospho-β-catenin (1:1000 dilution, S552), Non-phospho (active) β-catenin (1:1000, 8814 and 4270), Notch3 (1:1000, D11B8), Notch3 (1:1000, 8G5), β-actin total (1:1000), Oct4 (1:1000, 2750), Nanog (1:1000, 4903) were purchased from Cell Signaling Technologies. Hey1 was purchased from Themofisher Scientific (1:1000, PA5-23484). cMyc antibodies were obtained from Sigma (1:1000, M-4439). HDAC2 antibody was purchased from Santa Cruz Biotechnology (1:1000, SC-6296).
Arasada R.R., Shilo K., Yamada T., Zhang J., Yano S., Ghanem R., Wang W., Takeuchi S., Fukuda K., Katakami N., Tomii K., Ogushi F., Nishioka Y., Talabere T., Misra S., Duan W., Fadda P., Rahman M.A., Nana-Sinkam P., Evans J., Amann J., Tchekneva E.E., Dikov M.M, & Carbone D.P. (2018). Notch3-dependent β-catenin signaling mediates EGFR TKI drug persistence in EGFR mutant NSCLC. Nature Communications, 9, 3198.
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2

Western Blot Analysis of Protein Expression

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Total cell lysates were prepared in a Tris pH 6.8–10% SDS solution (1:1) by heating at 95° for 20 mins. Protein concentration was measured using Pierce BCA protein assay kit as per the company instructions. For western blotting 10–40 ug of protein was resolved on 7.5% or 10% mini gels from Biorad, transferred to nitrocellulose membrane using semi-dry method and immunoblotted. 10% BSA was used for filter blocking in all conditions.
The following primary antibodies were used: against Notch1, Slug and Actin (Santa Cruz Biotechnology), against PlexinD1 (R&D Systems), against Vinculin (Sigma), against Notch3 (Cell Signaling technology), against E-cadherin (BD Transduction laboratories).
Rehman M., Gurrapu S., Cagnoni G., Capparuccia L, & Tamagnone L. (2016). PlexinD1 Is a Novel Transcriptional Target and Effector of Notch Signaling in Cancer Cells. PLoS ONE, 11(10), e0164660.
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3

Notch Pathway and Epithelial-Mesenchymal Transition Antibodies

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Polyclonal antibodies to Notch1, Notch2, Notch3, Notch4, Dll1, Dll3, Dll4, Jagged1, Jagged2, HIF‐1α, N‐cadherin, E‐cadherin and Snail1 were purchased from Cell Signaling Technology (Beverly, MA). Polyclonal antibodies to G6PD, transketolase (TKT), Nrf2, Keap1 and monoclonal antibody to β‐actin were purchased from Abcam (Cambridge, UK). The dual‐luciferase reporter assay kit was obtained from Promega (Madison, WI).
Zhang H., Zhang Z., Du G., Sun H., Liu H., Zhou Z., Gou X., Wu X., Yu X, & Huang Y. (2019). Nrf2 promotes breast cancer cell migration via up‐regulation of G6PD/HIF‐1α/Notch1 axis. Journal of Cellular and Molecular Medicine, 23(5), 3451-3463.
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4

Antibody-based Protein Analysis and Chromatin Immunoprecipitation

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The antibodies used for immunoblotting were: p53 (DO-1, Santa Cruz, Cat# sc-126, at 1:1000 dilution), CBFB (Bethyl, Cat# A303-547A, at 1:1000 dilution), RUNX1 (Cell Signaling, Cat# 4334s, at 1:1000 dilution), NOTCH3 (Cell Signaling, Cat# 5276s, at 1:1000 dilution), TAp73 (Novus Biologicals, Cat# NBP2-24737, at 1:1000 dilution), ΔNp73 (Novus Biologicals, Cat#NBP2-24873, at 1:1000 dilution), and β-actin (Sigma, Cat# A5316, at 1:5000 dilution). The antibodies used for chromatin immunoprecipitation (ChIP) were: p53 (DO-1 Santa Cruz, Cat# sc-126, 10 μg) and RUNX1 (Abcam, Cat# ab23980, 10 μg).
Malik N., Yan H., Yang H.H., Ayaz G., DuBois W., Tseng Y.C., Kim Y.I., Jiang S., Liu C., Lee M, & Huang J. (2021). CBFB cooperates with p53 to maintain TAp73 expression and suppress breast cancer. PLoS Genetics, 17(5), e1009553.
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5

Notch Signaling Pathway Protein Analysis

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Protein extracts preparation was described elsewhere66 (link). Blots were incubated with primary antibodies against: Notch1Val1744 (2421; Cell Signaling Technology, Beverly, MA, USA), Notch3 (2889; Cell Signaling Technology, Beverly, MA, USA), Notch1 (sc-6014-R; Santa Cruz Biotechnology, Santa Cruz, CA, USA), Cyclin D3 (sc-182; Santa Cruz Biotechnology, Santa Cruz, CA, USA), CDK4 (sc-260; Santa Cruz Biotechnology, Santa Cruz, CA, USA), p27 (3688; Cell Signaling Technology, Beverly, MA, USA), PARP (9542; Cell Signaling Technology, Beverly, MA, USA) and β-actin (sc-47778; Santa Cruz Biotechnology, Santa Cruz, CA, USA); followed by hybridization with Antibodies HRP conjugated: anti‐rabbit (sc-2004; Santa Cruz Biotechnology, Santa Cruz, CA, USA), or anti-mouse (sc-2005; Santa Cruz Biotechnology, Santa Cruz, CA, USA).
Mori M., Tottone L., Quaglio D., Zhdanovskaya N., Ingallina C., Fusto M., Ghirga F., Peruzzi G., Crestoni M.E., Simeoni F., Giulimondi F., Talora C., Botta B., Screpanti I, & Palermo R. (2017). Identification of a novel chalcone derivative that inhibits Notch signaling in T-cell acute lymphoblastic leukemia. Scientific Reports, 7, 2213.
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6

Western Blot Analysis of Notch Pathway

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Pellets were processed and protein was extracted as previously described (34 (link)). Forty μg of lysate for each sample per lane was run in precast NuPAGE Novex polyacrylamide gel 4% to 12% Bis-Tris Gels (1.0-mm thick, 10-well) in 1X Tris-glycine (Life Technologies, Grand Island, NY). Lysates were transferred onto a nitrocellulose membrane (Bio-Rad, Hercules, CA), blocked in PBS containing 5% bovine serum albumin or nonfat dry milk powder, and incubated overnight with antibodies per manufacturer's directions. Blots were then washed several times with PBS containing 0.1% Tween 20 and incubated in peroxidase-conjugated immunoglobulin G diluted 1:5000 in blocking solution. After washing several times in PBS with 0.1% Tween 20, blots were developed with enhanced Western Lightning chemiluminescence reagent (Perkin Elmer, Waltham, MA) and exposed to film. Primary antibodies included HES1 (1:800 Aviva, San Diego, CA), NOTCH 3 (1:800, Cell Signaling Technologies, Inc.), JAG1 (1:800, Cell Signaling Technologies Inc.), DLL-1 (1:800 Cell Signaling Technologies, Inc.), glyceraldehyde 3-phosphate dehydrogenase ([GAPDH]; 1:5000, RDI, Flanders, NJ), and β-actin (1:500, mouse, Santa Cruz Biotechnology, Inc., Dallas, TX). Omission of primary antibody was used as a negative control.
Taylor I.C., Hütt-Cabezas M., Brandt W.D., Kambhampati M., Nazarian J., Chang H.T., Warren K.E., Eberhart C.G, & Raabe E.H. (2015). Disrupting NOTCH Slows Diffuse Intrinsic Pontine Glioma Growth, Enhances Radiation Sensitivity, and Shows Combinatorial Efficacy with Bromodomain Inhibition. Journal of neuropathology and experimental neurology, 74(8), 778-790.
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7

Immunoblotting for Protein Expression

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Total protein extract for each cell line was obtained by using a lysis buffer as described previously (Yang et al., 2010 (link)), and equal amounts (20 μg per load) were analyzed by immunoblotting. Antibody against β-actin was from Sigma-Aldrich (St. Louis, MO, United States) (A5441, 1:20,000); vascular endothelial growth factor (VEGF; sc-507, 1:1,000) was from Santa Cruz Biotechnology (Dallas, TX, United States). AKT (no. 9272, 1:1,000), extracellular signal-regulated kinase 1/2 (ERK1/2; mAb no. 4695, 1:1,000), and thrombospondin-1 (TSP-1) were from Lab Vision (MS-418, 1:500; Thermo Fisher Scientific, Waltham, MA, United States). Notch3 was from Cell Signaling Technology (Danvers, MA, United States) (cst-5276, 1:1,000). Western blotting reagents were from a chemiluminescence kit (Amersham Biosciences, Little Chalfont, United Kingdom).
Ji Z., Tian W., Gao W., Zang R., Wang H, & Yang G. (2021). Cancer-Associated Fibroblast-Derived Interleukin-8 Promotes Ovarian Cancer Cell Stemness and Malignancy Through the Notch3-Mediated Signaling. Frontiers in Cell and Developmental Biology, 9, 684505.
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8

NOTCH1 and NOTCH3 Knockdown in Cell Lysates

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Three different small interfering RNAs (siRNAs) targeting NOTCH1 (cat#4851-1, 4851-2, and 4851-3) and NOTCH3 (cat#4854-1, 4854-2, and 4854-3) were purchased from Bioneer (Deajeon, Korea). Non-targeting siRNA was used as a negative control. The RNAi oligonucleotide or RNAi negative control was transfected into the cells using a Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Cells were lysed with a cell extraction buffer (Thermo Fisher Scientific) containing protease inhibitors. Protein concentrations were determined using the BCA protein assay (Thermo Fisher Scientific). Protein samples were separated using SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were incubated with the indicated primary antibodies overnight at 4 °C. The following antibodies were used: NOTCH1 (cat#4380, Cell Signaling Technology, Danvers, MA, USA), NOTCH3 (cat#5276, Cell Signaling Technology), and α-tubulin (cat#sc-8035, Santa Cruz Biotechnology, Inc., Dallas, TX, USA). The blots were visualized using the Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific, Waltham, MA, USA). The original WB can be found at Figure S5.
Jeong S., Park S., Jo Y.S., Choi M.J., Lee G., Lee S.G., Choi M.C., Park H., Joo W.D., Jung S.G, & Lee J. (2022). Long Non-Coding RNA-Based Functional Prediction Reveals Novel Targets in Notch-Upregulated Ovarian Cancer. Cancers, 14(6), 1557.
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9

Simple Western Protein Analysis

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Protein lysates from tissue and in vitro cell culture lines were processed for use on Wes Simple Western assays according to manufacturer protocols (Protein Simple). Samples were mixed with Simple Western Sample Master Mix (80 mmol/L DTT, 2× sample buffer, 2× fluorescence standard) and denatured. The Simple Western kit plate was loaded with denatured samples, primary antibody, HRP‐conjugated anti‐rabbit antibody, luminol‐peroxide substrate and wash buffers. The proprietary capillary‐based separation system was utilized to automatically load, separate, immobilize and immunoprobe protein lysates for proteins of interest using HRP‐mediated chemiluminescence. The chemiluminescent signal was detected using the system's built‐in CCD camera and analysed for signal intensity using accompanying Compass software. Band intensity was used to generate a traditional Western blot lane. Primary antibodies were titrated to determine optimal protein and primary antibody concentration. Antibodies for pERK1/2 (1:100 dilution, rabbit monoclonal, #4695), pSMAD2/3 (1:50 dilution, rabbit monoclonal, #8828), vinculin (1:150 dilution, rabbit polyclonal #4650) and Notch3 (1:100 dilution, rabbit monoclonal #5276) were purchased from Cell Signaling Technology.
Pedroza A.J., Koyano T., Trojan J., Rubin A., Palmon I., Jaatinen K., Burdon G., Chang P., Tashima Y., Cui J.Z., Berry G., Iosef C, & Fischbein M.P. (2019). Divergent effects of canonical and non‐canonical TGF‐β signalling on mixed contractile‐synthetic smooth muscle cell phenotype in human Marfan syndrome aortic root aneurysms. Journal of Cellular and Molecular Medicine, 24(3), 2369-2383.
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10

In vitro and in vivo BET inhibitor evaluation

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For in vitro experiments, we used the BETi CPI203, purchased from Sigma-Aldrich (#SML1212), and the BETi CN210, kindly provided by ConverGene. The inhibitors were re-suspended in dimethyl sulfoxide (DMSO; #D2650; Sigma-Aldrich) to make a working stock of 10 mM. DMSO diluted at 1:1,000 was used as the vehicle control for all in vitro experiments. For Western blots, we used the following antibodies: Notch3 (#D11B8; Cell Signaling Technology; 1:1,000), Hes1 (#D6P2U; Cell Signaling Technology; 1:700), BRD4 (#A700–004; Bethyl Laboratories; 1:5,000), and vinculin (#V9131; Sigma-Aldrich; 1:10,000). For IHC analysis of paraffin sections, the following antibodies were used: BRD4 (#A700–004; Bethyl Laboratories; 1:100), Notch3 (#ab23426; Abcam; 1:100), Ki67 (#RB-9043-P1; NeoMarkers, 1:200), and cleaved caspase-3 (#9661S; Cell Signaling Technology; 1:100). For doxycycline induction of shRNA in vitro, we treated cells with 100 ng/mL of doxycycline (#D9891; Sigma-Aldrich). For in vivo induction of BRD4 shRNA, mice harboring OVCAR 5 tumors were placed on a daily 200 mg/kg doxycycline-chow diet (#14727450; Fisher Scientific).
Villar-Prados A., Wu S.Y., Court K.A., Ma S., LaFargue C., Chowdhury M.A., Engelhardt M.I., Ivan C., Ram P.T., Wang Y., Baggerly K., Rodriguez-Aguayo C., Lopez-Berestein G., Ming-Yang S., Maloney D.J., Yoshioka M., Strovel J.W., Roszik J, & Sood A.K. (2018). Predicting novel therapies and targets: regulation of Notch3 by the bromodomain protein BRD4. Molecular cancer therapeutics, 18(2), 421-436.
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