Anti notch1

Manufactured by Cell Signaling Technology

Sourced in United States, Macao

Anti-Notch1 is a primary antibody product manufactured by Cell Signaling Technology. It is designed to detect Notch1, a transmembrane protein that plays a crucial role in cell-cell communication and development.

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Lab products found in correlation

47 protocols using anti notch1

Immunoblotting of Cellular Signaling Proteins

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Total lysates prepared from subconfluent cells were immunoblotted with anti-MUC1-C (HM-1630-P1ABX, 1:400 dilution; Thermo Fisher Scientific), anti-JUN (3742, 1:1,000; Cell Signaling Technology), anti-ARID1A (12354, 1:1,000; Cell Signaling Technology), anti-NOTCH1 (3608, 1:1,000; Cell Signaling Technology), anti-β-actin (A5441, 1:100,000; Sigma), and anti-GAPDH (5174, 1:5,000; Cell Signaling Technology).

Bhattacharya A., Fushimi A., Yamashita N., Hagiwara M., Morimoto Y., Rajabi H., Long M.D., Abdulla M., Ahmad R., Street K., Liu S., Liu T, & Kufe D. (2022). MUC1-C Dictates JUN and BAF-Mediated Chromatin Remodeling at Enhancer Signatures in Cancer Stem Cells. Molecular Cancer Research, 20(4), 556-567.

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Characterization of Bone Marrow LSK Cells

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Bone marrow cells from untreated animals were collected as described above. First, we measured the expression of FPR2 in LSK cells. The anti-FPR2-FITC antibody was used (Bioss, Massachusetts, USA) to quantify this expression by flow cytometry. Afterwards, bone marrow cells were treated with rAnxA1 (100 nM) for 12 h, and viability of the LSK population determined using 7-aminoactinomycinD (7-AAD; Sigma Aldrich, USA); cell cycle phases and expression of Ki67 and Notch-1 were also evaluated. These quantifications were carried out by flow cytometry. Cells were fixed in 2% paraformaldehyde for 30 min, washed with 0.1 M glycine, and permeabilized with 0.001% Triton X-100. Subsequently, 2 × 10⁶ cells were incubated with primary anti-cyclin B1, anti-Notch-1 or anti-Ki67 (Cell Signaling Technology) antibodies for 2 h, and then 40 min with secondary rabbit Anti-IgG Alexa Fluor 488 (Molecular Probes/Invitrogen, USA). Analyses were performed on the LSK population gated as described above, using an Accuri C6 flow cytometer (Becton Dickinson, USA) and the FlowJo software.

Barbosa C.M., Fock R.A., Hastreiter A.A., Reutelingsperger C., Perretti M., Paredes-Gamero E.J, & Farsky S.H. (2019). Extracellular annexin-A1 promotes myeloid/granulocytic differentiation of hematopoietic stem/progenitor cells via the Ca2+/MAPK signalling transduction pathway. Cell Death Discovery, 5, 135.

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Immunohistochemical Analysis of Mouse Tumor

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The tumor formed in the mouse model was used for the IHC staining study. The staining assay was performed according to the standard protocol on five-micrometer formalin-fixed paraffin sections. After staining, a pathologist and an investigator blind to the study design scored the staining intensities according to an immunoreactive score (IRS) system [14 (link)]. The primary antibodies for IHC are anti-Nrf2 (ABclonal, 1 : 200), anti-Ki-67 (Santa Cruz, 1 : 200), anti-Notch1 (Cell Signaling, 1 : 100), anti-c-Myc (Santa Cruz, 1: 100), and anti-Slug (Santa Cruz, 1 : 100).

Gong Z., Xue L., Wei M., Liu Z., Vlantis A.C., van Hasselt C.A., Chan J.Y., Li D., Zeng X., Tong M.C, & Chen G.G. (2021). The Knockdown of Nrf2 Suppressed Tumor Growth and Increased the Sensitivity to Lenvatinib in Anaplastic Thyroid Cancer. Oxidative Medicine and Cellular Longevity, 2021, 3900330.

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Western Blot Analysis of Protein Markers

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Protein concentrations were determined with a BCA protein assay kit (Beyotime, P0012i). Twenty to thirty micrograms of total protein was separated by 10% SDS-PAGE (EpiZyme, pg112) and then transferred onto nitrocellulose membranes (Pall, 66485). Nonspecific sites were blocked with 5% nonfat milk in phosphate-buffered saline (with 0.1% Tween-20) at room temperature. Next, the blots were incubated overnight at 4 ℃ with the following primary antibodies: anti-Hbα (Santa Cruz, sc-514378), anti-Hbβ (Santa Cruz, sc-21757), anti-CD31 (Affinity, AF6191), anti-glutathione peroxidase 4 (GPX4) (Affinity, DF6701), anti-heme oxygenase-1 (HO-1) (Affinity, AF5393), anti-Bcl2 (ImmunoWay, YM3041), anti-Bax (ImmunoWay, YT0455), anti-Notch1 (Cell Signaling Technology, #3608), anti-Jag1 (Cell Signaling Technology, #70109), anti-Hes1 (Abcam, ab108937), anti-Hey1 (Abcam, ab154077), and anti-β-actin (Affinity, AF7018). The next day, the blots were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Affinity, s0001) and visualized with New Super ECL assay kit (KeyGen BioTECH, KGP1128).

Shan L., Xu X., Zhang J., Cai P., Gao H., Lu Y., Shi J., Guo Y, & Su Y. (2021). Increased hemoglobin and heme in MALDI-TOF MS analysis induce ferroptosis and promote degeneration of herniated human nucleus pulposus. Molecular Medicine, 27, 103.

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Western Blot Analysis of Notch Signaling

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Western blotting was performed as described previously (17 (link)). The primary antibodies and their sources were as follows: anti-Notch1, anti-Hes1, anti-NICD, anti-pERK, anti-ERK, and anti-pAKT (Cell Signaling Technology, Beverly, MA, USA); anti-Jagged1, anti-β-actin, anti-AKT, and anti-DUSP1 (Santa Cruz Biotechnology); anti-HBx (Merck-Millipore); anti-PTEN (Proteintech Group, Chicago, IL, USA). Horseradish peroxidase-conjugated secondary antibodies were purchased from Jackson Immuno Research Laboratories (West Grove, PA, USA).

Liao B., Zhou H., Liang H, & Li C. (2017). Regulation of ERK and AKT pathways by hepatitis B virus X protein via the Notch1 pathway in hepatocellular carcinoma. International Journal of Oncology, 51(5), 1449-1459.

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Western Blot Analysis of Notch Signaling

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Anti-Actin (catalog number [Cat#] sc-47778, 1:2,000), anti-p130 (Cat# sc-374521, 1:3,000), anti-p21 (Cat# sc-53870, 1:3,000), anti-p130 (Cat# sc-374521, 1:3,000), and anti-PML (Cat# sc-377390, 1:3,000) were purchased from Santa Cruz Biotechnology. Anti-TAp73 (Cat# A300-126A, 1:1,000) was purchased from Bethyl Laboratories, Inc. Anti-HA (Cat# 901513, 1:2,000) was purchased from BioLegend. Anti-Cleaved Notch1 (Cat# 4741, 1:1,000), anti-Notch1 (Cat# 4380T, 1:1,000), and anti-Hes1 (Cat# 11988S, 1:800) were purchased from Cell Signaling Technology. WesternBright ECL HRP substrate (Cat# K-12043-D20) was purchased from Advansta. Scrambled siRNA (5′-GGC CGA UUG UCA AAU AAU U-3′), sip73α1 siRNA (5′-ACC UGG GGC CCG UGG UUU-3′), siE11 siRNA#1 (5′-GCA CAG UUC GGC AGC UAC A-3′), siE11 siRNA#2 (5′-UCC UCU CGC CCA UGA ACA A-3′), and siNotch1 siRNA (5′-ACA AAG AUA UGC AGA ACA A-3′) were purchased from Horizon Discovery Biosciences Limited. RNAiMax (Cat# 13778150, Invitrogen), Protease Inhibitor Mixture (Cat# 78438), Magnetic Protein A/G beads (Cat# 78609), RevertAid RT Reverse Transcription Kit (Cat# K1691), and DreamTaq DNA Polymerase (Cat# EP0702) were all purchased from ThermoFisher. All reagents were used according to the manufacturer’s protocol.

Laubach K.N., Yan W., Kong X., Sun W., Chen M., Zhang J, & Chen X. (2022). p73α1, a p73 C-terminal isoform, regulates tumor suppression and the inflammatory response via Notch1. Proceedings of the National Academy of Sciences of the United States of America, 119(22), e2123202119.

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Characterizing EMT Pathway Components

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We performed this experiment as standard procedure in accordance with previous descriptions [8 (link)]. The following antibodies were used: anti-E-cadherin (cell signaling technology, USA), anti-N-cadherin (cell signaling technology, USA), anti-Snail (cell signaling technology, USA), anti-MMP-9 (cell signaling technology, USA), anti-Numb (cell signaling technology, USA), anti-Notch1 (cell signaling technology, USA), anti-FAK (cell signaling technology, USA), anti-p-FAK (cell signaling technology, USA), anti-PTEN (cell signaling technology, USA), anti-RBP-Jκ (millipore, USA), anti-GAPDH (Proteintech, USA).

Li J.Y., Huang W.X., Zhou X., Chen J, & Li Z. (2019). Numb inhibits epithelial-mesenchymal transition via RBP-Jκ-dependent Notch1/PTEN/FAK signaling pathway in tongue cancer. BMC Cancer, 19, 391.

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Antibody Characterization for Western Blot and Immunostaining

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Antibodies and reagents were obtained as follows: anti-Notch1 (western blot (WB), 1:800; immunohistochemical (IHC) or immunofluorescence (IF) staining, 1:400), anti-E-cadherin (WB and IF, 1:400), anti-N-cadherin (WB, 1:400), anti-α-SMA (WB, 1:800, IHC and IF, 1:400), and anti-Ki67 (IHC and IF, 1:400) antibodies from Cell Signalling Technology (CST, Beverly, MA, USA); anti-NICD (WB, 1:400, IHC and IF, 1:200), anti-Histone H3 (WB, 1:800), anti-c-Myc (WB, 1:800), and anti-vimentin (WB, 1:800; IHC, 1:400) antibodies from Abcam Company (Cambridge, MA, USA); anti-Jagged1 (WB, 1:800) antibody from Santa Cruz Biotechnology; anti-Smad2/Smad3 (phospho T8) (WB, 1:800) and anti-TGF-β1R (WB, 1:800) antibodies from MDL Biotechnology (Beijing, China); anti-GAPDH (WB, 1:8000), anti-Smad2/3 (WB and IF, 1:1000), anti-p-Smad2 (WB, 1:1000), anti-p-Smad3 (WB, 1:1000), anti-c-Myc (WB, 1:800); anti-collagen I (WB, 1:800; IF, 1:200), and anti-collagen III (WB, 1:800, IHC and IF, 1:200) antibodies from Biogot Technology (Shanghai, China); and anti-TGF-β1 (WB, 1:800) and anti-β-actin (WB, 1:8000) antibodies from Proteintech Biotechnology (Wuhan, China).

Hong W., Zhang G., Lu H., Guo Y., Zheng S., Zhu H., Xiao Y., Papa A.P., Wu C., Sun L., Chen B, & Bai Y. (2019). Epithelial and interstitial Notch1 activity contributes to the myofibroblastic phenotype and fibrosis. Cell Communication and Signaling : CCS, 17, 145.

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Immunofluorescence Analysis of Kidney Markers

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Immunofluorescent microscopy was performed using 8 μm paraffin sections washed in xylene and re-hydrated in ethanol. Sections were heated in a pressure cooker in 0.1 M sodium citrate buffer (pH 6.0), blocked in a 30% BSA/donkey serum solution, and incubated with primary antibodies overnight at 4 °C. Tissue was incubated with either Alexa Fluor 488/594 goat-anti-mouse or anti-rabbit secondary antibodies (1:500, Invitrogen) and DAPI for 1 h at room temperature, mounted in VectaShield (Vector Labs, CA), and visualized on a Leica DM2500 fluorescent microscope. The primary antibodies that were utilized were: anti-Cited1 (1:200, NeoMarkers, MI), anti-Jagged1 (1:100, Santa Cruz, TX) anti- Notch1 (1:100, Cell Signaling, MA), anti-NCAM (1:200, Sigma, MO), anti-Six2 (1:200, Proteintech, IL), and anti-Frs2α 1: 1:100 (Abcam, MA). DBA and LTL lectins were used at a dilution of 1:200 (Vector Labs, CA).

Di Giovanni V., Walker K.A., Bushnell D., Schaefer C., Sims-Lucas S., Puri P, & Bates C.M. (2015). Fibroblast growth factor receptor–Frs2α signaling is critical for nephron progenitors. Developmental biology, 400(1), 82-93.

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Western Blot Analysis of Protein Markers

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Proteins were exacted with an appropriate volume of lysis buffer. After electrophoresis on SDS-PAGE, proteins were transferred onto PVDF membranes (Millipore). Antibodies used here were anti-LC3I/II (Sigma), anti-P62 (Proteintech), anti-notch1 (Cell Signaling Technology), anti-Hes-1 (Santa Cruz), anti-GAPDH (Cell Signaling Technology), anti-Tublin (Cell Signaling Technology) and goat anti-rabbit IgG HRP (Cell Signaling Technology).

Li X., Liu F., Zhang X., Shi G., Ren J., Ji J., Ding L., Fan H., Dou H, & Hou Y. (2016). Notch-Hes-1 axis controls TLR7-mediated autophagic death of macrophage via induction of P62 in mice with lupus. Cell Death & Disease, 7(8), e2341-.

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