Protein lysates in NuPAGE buffer (Invitrogen) were separated by gradient 4–12% BisTris Gel and transferred to polyvinylidene difluoride membrane. Primary antibodies used in these experiments included rabbit monoclonal anti-Notch1 (D1E11; 1:1000; Cell Signaling, Danvers, MA, USA), rabbit polyclonal anti-cleaved Notch1 (Val1744; 1:1000; Cell Signaling), rabbit monoclonal anti-Notch2 (8A1; 1:1000; Cell Signaling), rabbit polyclonal anti-Notch3 (Pro2311; 1:1000; Cell Signaling), rabbit polyclonal anti-HES1 (ab71559; 1:1000; Abcam, Cambridge, MA, USA), rabbit polyclonal anti-HEY1 (ab22614; 1:500; Abcam), rabbit polyclonal anti-HES6 (ab66461; 1:1000; Abcam) and mouse monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (clone 6C5; 1:1000; Santa Cruz Biotechnology, Dallas, Tx, USA). The membrane was incubated sequentially with primary antibodies (overnight at 4°C), horseradish peroxidase-conjugated anti-rabbit or antimouse secondary antibodies, and chemiluminescent substrate (Thermo Fisher, Waltham, MA, USA) before exposure to film.
Val1744
Manufactured by Cell Signaling Technology
Sourced in United States
Val1744 is a protein that functions as a tyrosine kinase inhibitor. It specifically targets the NOTCH1 receptor and blocks its signaling pathway.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 700, by Zeiss (2 mentions)
Ab71559, by Abcam (1 mentions)
Ab22614, by Abcam (1 mentions)
Ab66461, by Abcam (1 mentions)
Horseradish peroxidase conjugated anti rabbit or anti mouse secondary antibodies, by Thermo Fisher Scientific (1 mentions)
Chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Notch1, by Santa Cruz Biotechnology (1 mentions)
Jagged1, by Santa Cruz Biotechnology (1 mentions)
β actin, by Merck Group (1 mentions)
Notch1, by Abcam (1 mentions)
Sc 28310, by Santa Cruz Biotechnology (1 mentions)
Ventana benchmark xt, by Roche (1 mentions)
Tunicamycin, by Fujifilm (1 mentions)
Alexa fluor 488 conjugated anti mouse igg antibody, by Thermo Fisher Scientific (1 mentions)
Neun a60, by Merck Group (1 mentions)
Horseradish peroxidase hrp conjugated anti mouse and anti rabbit igg antibodies, by GE Healthcare (1 mentions)
Alexa fluor 546 conjugated anti rabbit igg antibody, by Thermo Fisher Scientific (1 mentions)
Western lightning chemiluminescence reagent plus, by PerkinElmer (1 mentions)
Ab58310, by Abcam (1 mentions)
Mu392 uc, by BioGenex (1 mentions)
12 protocols using val1744
1
Notch Signaling Pathway Protein Analysis
2
Antibodies for V-ATPase Subunits Analysis
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Antibodies raised against the V-ATPase ‘a’ subunit isoforms a1 and a2 were generated in our laboratory. The mouse anti-a2V neutralizing antibody against 488–510 amino acids of trans-membrane region (Antibody clone 2C1) and rabbit anti-a2NTD against N terminal domain (Antibody Clone 470) were used as described previously [25 (link), 44 (link), 60 (link)]. Anti a1 antibody was raised in rabbit against the synthetic peptides from unique regions of a1 (amino acids 73–95; RKANIPIMDTGENPEVPFPRD) by Covance (USA) and anti a3 antibody was purchased from Abnova, USA. Notch1 (antibody clone EP1238Y) and organellar markers Rab5, Pan-Cadherin and Golph4 were from Abcam. For Western Blot we used cleaved Notch1 antibody Val1744 (Cell signaling, Danvers, MA), Jagged1 (Antibody clone H114, Santa-Cruz, CA), LC3B (Abcam). β-actin (antibody clone AC-74) was purchased from Sigma Aldrich and used as the loading control. For immunohistochemistry we used Notch-1(Santa-Cruz, antibody clone C-20), and anti-a2V. For flow cytometry we used Notch1-APC (Biolegend, San Diego, CA) and FITC conjugated anti-a2V (Covance, Princeton, NJ).
3
Immunohistochemical Profiling of Breast Cancer
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Formalin‐fixed paraffin‐embedded BC specimens were retrieved from the archives of the Institute of Pathology. Specimens of the RC cohort underwent an expert histopathological review regarding tumor type, stage, and grade. One millimeter cores of the periphery and the tumor center were assembled in triplicate in a tissue‐microarray (TMA), and hematoxylin‐eosin stained sections were used as templates. Immunohistochemical protein detection on 5 µm deparaffinized sections of primary BCs, xenograft tumors, or embedded cell lines was performed with an ultraView Universal 3,3′‐diaminobenzidine (DAB) detection kit on a Ventana Benchmark XT autostainer (Ventana Medical Systems). For cleaved NOTCH1 (N1ICD), the primary rabbit anti‐human antibody Val1744 (Cell Signaling) was used at 1:100 dilution and for fos‐related antigen 1 (FRA1), primary mouse anti‐human antibody sc‐28310 (Santa Cruz) was used at 1:50 dilution. Staining for both N1ICD and FRA1 was assessed blinded from clinical outcome. Tumors were given scores of absent (0), weak (1), moderate (2) or strong (3) staining. Weak and absent staining then were defined as low expression, whereas moderate and strong staining were defined as high expression.
4
Neuronal Differentiation Regulation by Endoplasmic Reticulum Stress
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Tunicamycin was purchased from Wako Pure Chemical Industries (Osaka, Japan). 2-Deoxy-D-glucose and rabbit polyclonal antibodies against glial fibrillary acidic protein (GFAP), doublecortin (DCX), and HRD1 (C-terminal) were purchased from Sigma-Aldrich (St. Louis, MO). Mouse monoclonal antibodies against nestin, βIII-tubulin, neuronal nuclei protein (NeuN; A60), and microtubule-associated protein-2 (MAP-2) were purchased from Millipore (Temecula, CA). A mouse monoclonal antibody against KDEL (10C3) was purchased from Stressgen Biotechnologies (Victoria, British Columbia, Canada). A rabbit polyclonal antibody against cleaved Notch1 (Val1744) was purchased from Cell Signaling Technology (Danvers, MA). A goat polyclonal antibody against Notch3 (M-20) and a rabbit polyclonal antibody against GADD 153 (CHOP) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase (HRP)-conjugated anti-mouse and anti-rabbit IgG antibodies were purchased from GE Healthcare (Buckinghamshire, United Kingdom). An Alexa Fluor 488-conjugated anti-mouse IgG antibody and an Alexa Fluor 546-conjugated anti-rabbit IgG antibody were purchased from Life Technologies (Grand Island, NY). Western Lightning Chemiluminescence Reagent Plus was obtained from PerkinElmer Life Science (Waltham, MA).
5
Immunohistochemistry of Notch Signaling
6
Intracellular Notch1 Level Quantification
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The intracellular Notch 1 level was determined by Western blot analysis. Pro–B cells were cultured on a monolayer for OP9DL1 stroma cells for 24 h and the stimulation was terminated by placing the cells on ice. The pro–B cells were removed carefully without disturbing the OP9DL1 stroma cells, centrifuged, and lysed in RIPA buffer supplemented with Protease inhibitor (Roche; 11697498001) and PMSF (1 µM; Sigma-Aldrich; P7626). Protein concentration was determined by Bradford reagent (Sigma-Aldrich; B6916), 50 µg of protein from each sample was boiled for 5 min in Laemmli buffer, and the proteins were separated on SDS-PAGE (456–8081; Bio-Rad Laboratories). The proteins were electrophoretically transferred to PVDF membranes (Immobulin FL; Millipore). Membranes were blocked with 5% BSA and probed with antibodies against intracellular Notch1 (Val1744; Cell Signaling Technology; 2421; 1:250 dilution), and equal loading was determined by probing blots with antibodies against GAPDH (ab9483; Abcam). Proteins were detected by IR dyes (Odyssey) and the membrane were scanned using Odyssey CLx Infrared Imaging System.
7
Notch Signaling Regulation by Lipid Mediators
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Endothelial cells were seeded onto 6-well plates coated with or without 500 ng/ml recombinant mouse Dll4 protein (R&D Systems) in 0.1% BSA/PBS for 1 h at room temperature. Experiments were performed in the presence of solvent, 10 µmol/liter 19,20-EDP/DHDP, 10 µmol/liter 11,12-EET/DHET, 10 µmol/liter 14,15-EET/DHET, 3 µmol/liter 12,13-EpOME/DiHOME, 20 µmol/liter DAPT, or 10 µmol/liter MG 132. After an additional 4 h, cells were washed three times with PBS and lysed in a Triton X-100–containing buffer. Intracellular domain of Notch (NICD) levels were assessed by Western blotting with an S3 (γ-secretase) cleavage-specific antibody Val1744 (Cell Signaling Technology). For the nuclear translocation of NICD, cells were fixed with 4% PFA for 15 min at room temperature, followed by NICD and DAPI immunostaining. Co-localization of NICD with DAPI was quantified with ImageJ software (National Institutes of Health).
8
Quantifying NOTCH1 Signaling and Expression
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Western blots were stained with antibodies specific for γ-secretase–cleaved NOTCH1 (Val1744; #4147 or #2421; Cell Signaling Technology), the intracellular transcriptional activation domain of NOTCH1 (Hasserjian et al., 1996 (link)), or the C terminus of NOTCH1 (#SC-6014 [C-20]; Santa Cruz Biotechnology). Control stains were performed with antibodies specific for actin (#ACTN05; Thermo Fisher Scientific), vinculin (#2907; Abcam), or GAPDH (#137179; Santa Cruz Biotechnology). Blots were developed with anti-rabbit HRP (#NA9340V; Amersham) or anti-mouse-HRP (#NA9310V; Amersham).
Expression of FR1 and FR2 was determined using isoform-specific antibodies (#MAB5646, R&D Systems; #103988, Abcam). Immunofluorescence staining was performed on permeabilized cells using a murine monoclonal antibody against NOTCH1 (3294, Abcam), and species-specific secondary antibodies linked to Alexa Fluor 488. Slides were mounted with Prolong Gold anti-fade reagents and counterstained with DAPI (Invitrogen). Images were acquired using a Zeiss LSM510 confocal microscope at 100× power. Cell surface NOTCH1 was evaluated by staining nonpermeabilized cells with monoclonal anti-human NOTCH1 antibody (#FAB5317P; R&D Systems) as previously described (Roti et al., 2013 (link)).
Expression of FR1 and FR2 was determined using isoform-specific antibodies (#MAB5646, R&D Systems; #103988, Abcam). Immunofluorescence staining was performed on permeabilized cells using a murine monoclonal antibody against NOTCH1 (3294, Abcam), and species-specific secondary antibodies linked to Alexa Fluor 488. Slides were mounted with Prolong Gold anti-fade reagents and counterstained with DAPI (Invitrogen). Images were acquired using a Zeiss LSM510 confocal microscope at 100× power. Cell surface NOTCH1 was evaluated by staining nonpermeabilized cells with monoclonal anti-human NOTCH1 antibody (#FAB5317P; R&D Systems) as previously described (Roti et al., 2013 (link)).
9
Western Blot Analysis of Notch Signaling
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Eighty μg of proteins for each sample were separated on a 4–12% SDS-polyacrylamide gel (Life Technologies, Monza, Italy) and transferred onto nitrocellulose membrane. After being blocked with nonfat dry milk, the membranes were incubated overnight at +4 °C with the following primary antibodies: mouse monoclonal anti-NOTCH1 1:1000 (clone mN1A, catalog number 629101, Biolegend, SA, USA), rabbit polyclonal anti-cleaved NOTCH1 1:500 (Val1744, catalog number 2421, Cell Signaling Technologies, Leiden, Netherlands), rabbit polyclonal anti-JAG1 1:200 (clone H-114, catalog number sc-8303, Santa Cruz Biotechnology, Heidelberg, Germany), rabbit monoclonal anti-ZEB1 (anti-TCF8/ZEB1, 1:1000, clone: D80D3, Cell Signaling), rabbit polyclonal anti-MMP9 (1:1000, catalog number: 3852, Cell Signaling), and rabbit monoclonal anti-CDH1 (1:1000, clone: 24E10, catalog number: 3852, Cell Signaling). Following incubation with the appropriate secondary antibody, the signal was detected with Chemiluminescent Sensitive Plus HRP (BioFx Laboratories, MD, United States), and images were acquired with the ChemidocTM XRS+ (Bio-Rad Laboratories). Mouse monoclonal anti-βACTIN (1:2000, clone AC-15, catalog number A1978, Sigma Aldrich) was used as housekeeping protein. Each experiment was repeated at least twice.
10
Modulation of Notch Signaling in Retinal ECs
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