NB-360, NB-449, NB-444, NB-480 and NB-109 are products of Novartis Pharma AG. The γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT) was purchased from Sigma (D5942). All compounds were dissolved in DMSO. Stocks of 50 mM solution were prepared and stored as aliquots in −20 °C to reduce several freeze thaw cycles.
N n 3 5 difluorophenacetyl l alanyl s phenylglycine t butyl ester dapt
Manufactured by Merck Group
Sourced in United States, Germany, Belgium
N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) is a chemical compound used in research. It functions as a gamma-secretase inhibitor.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Hif 1α, by Cell Signaling Technology (2 mentions)
Antibiotic antimycotic solution, by Corning (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Wortmannin, by Merck Group (2 mentions)
Opaque 96 well plate, by Corning (2 mentions)
Celltiter glo luminescent assay, by Promega (2 mentions)
Nb 360, by Novartis (1 mentions)
High glucose dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Accutase, by Merck Group (1 mentions)
Histone h3, by Cell Signaling Technology (1 mentions)
Cetuximab, by Merck Group (1 mentions)
P egfr tyr1068, by Cell Signaling Technology (1 mentions)
Vascular endothelial growth factor (vegf), by Cell Signaling Technology (1 mentions)
Mia paca 2, by American Type Culture Collection (1 mentions)
Bxpc 3, by American Type Culture Collection (1 mentions)
Panc 1, by American Type Culture Collection (1 mentions)
Sodium pyruvate, by Corning (1 mentions)
Gentamycin, by Corning (1 mentions)
27 protocols using n n 3 5 difluorophenacetyl l alanyl s phenylglycine t butyl ester dapt
1
Novartis Compounds for γ-Secretase Inhibition
2
Culturing HepG2 and HBV-Transfected Cells
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The human hepatoma cell line HepG2 and HBV genome-transfected HepG2 (HepG2.2.15) cells were obtained from the China Center for Type Culture Collection (Wuhan, China). Cells were cultured in high glucose Dulbecco's modified Eagle's medium (Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (Gibco). Dimethyl sulfoxide (DMSO) and γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). DAPT was dissolved in DMSO.
3
Modulation of NK Cell Function by MSCs
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UC-MSCs were seeded in 24- or 48-well plates and allowed to adhere. After 24 hours, freshly isolated, FACS-sorted NK cells from unrelated donors were added at a MSC:NK-ratio of 1:10. Following 16 hours of co-culture, the NK cells were removed from the adherent MSCs by pipetting for phenotyping or were tested for cytokine producing ability by intracellular staining or for cytotoxic potential against K562 leukemic cells using CD107a degranulation or chromium release assay. NK cells cultured without MSCs were used as controls. Cell-free supernatants from the NK-MSC co-cultures or MSC cultures were frozen for further analysis as MSC-NK-conditioned medium (NK-MSC cm) or MSC conditioned media (MSC cm), respectively. For determining the influence of conditioned media, NK cells from the unrelated donors were cultured for 16 hours in the cell-free supernatants generated as mentioned above. To determine the role of gamma-secretase activation, N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) (Sigma-Aldrich, Germany), a gamma-secretase inhibitor, was added to the co-cultures at 6 μM or 10 μM concentrations. DMSO was used as vehicle control. 5 μM NS-398 was added to the co-cultures to block the action of cyclooxygenase (COX)-2. IL-1β neutralising antibody or the matched isotype control antibody was added to the co-cultures at a concentration of 10 ng/ml.
4
Notch Inhibition in Retinal Neurospheres
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For Notch inhibition studies, prenatal retinal neurospheres were dissociated to single cell suspensions by addition of Accutase (Millipore, Temecula CA) for 10 min at 37°C. Cells (50,000 in 50 μl) were plated onto laminin/poly-L-ornithine-coated glass coverslips in standard medium for 24 hr, treated with either vehicle (0.2% dimethyl sulfoxide) (Sigma-Aldrich, St. Louis MO) or 10 μM N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S phenylglycine t-butyl ester (DAPT) (Sigma-Aldrich, St. Louis MO) in standard medium for 24 hr, washed 3 times to remove DAPT, and cultured for an additional 7 days in medium supplemented with 2% B27 only.
5
Investigating M2 Receptor Modulation in GBM Cells
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At 24 h from seeding, cells were incubated in the presence of the M2 muscarinic receptor agonist arecaidine propargyl ester (APE) (100 µM) (Sigma-Aldrich, St. Louis, MO, USA) for different time points according to the experimental plan (24 h, 48 h). The selective binding of APE to M2 receptors has been previously demonstrated by pharmacological competition binding assay and M2 silencing experiments both in the GBM cell lines and in GB cancer stem cells [18 (link),19 (link),34 (link)].
GBM cells were also incubated for 24 h with 5 µM N-[N-(3,5-difluorophenacetyl)-l -alanyl]-(S)-phenylglycine t-butyl ester (DAPT) (Sigma-Aldrich, St. Louis, MO, USA), a gamma-secretase inhibitor or 10 µM N-(3-Chlorophenyl)-6,7-dimethoxy-4-quinazolinamine (Tyrphostin; Tyrph) (Sigma–Aldrich, St. Louis, MO, USA), as an inhibitor of EGFR activity.
GBM cells were also incubated for 24 h with 5 µM N-[N-(3,5-difluorophenacetyl)-
6
Targeting EGFR and Notch1 Signaling
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All chemicals and reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA), unless indicated. Antibodies against EGFR, p-EGFRTyr1068, HIF-1α, and Notch1, Notch1 intracellular domain (NICD), Hes1, VEGF, Histone H3 were obtained from Cell Signaling Technologies (Danvers, MA, USA), CD31 were obtained from BD Pharmingen (NJ, USA). Cetuximab was purchased from Merck (Darmstadt, Germany). N-[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester (DAPT, γ-secretase inhibitor which inhibited cleavage of Notch1) was obtained from Sigma-Aldrich (St. Louis, MO, USA).
7
Culturing Pancreatic Cancer Cell Lines
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Human pancreatic cancer cells PanC-1, MiaPaCa-2, and BxPC-3 (all cell lines obtained from American Type Culture Collection, at passage 4) were grown in RPMI 1640 containing 10% heat inactivated fetal bovine serum (Sigma-Alrich, St. Louis, MO) and 1% antibiotic-antimycotic solution (Corning, Tewksbury, MA) at 37°C in a humidified atmosphere of 5% CO2. HPNE cells were kindly provided by Dr. Anirban Maitra, Johns Hopkins University School of Medicine and grown in DMEM with 4.5 g/L glucose, L-glutamine and Sodium Pyruvate (Corning, Tewksbury, MA) with 5% FBS, 1X N2, 10 ng/ml bFGF and 50 μg/ml Gentamycin. All the cell lines used in this study were within 20 passages after receipt or resuscitation (∼3 months of non-continuous culturing). The cell lines were not authenticated as they came from national repositories. Quinomycin was purchased from (AG Scientific, San Diego, CA) and our collaborator. N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) was purchased from (Sigma-Alrich, St. Louis, MO).
8
Isolation and Purification of TF3 Monomer
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TF3 monomer (purity: 92.4%) was isolated and purified using previously established method (15 (link)). Wortmannin and N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) were purchased from Sigma and Santa Cruz Biotechnology (Santa Cruz, CA, USA), respectively. Antibodies against phospho-Akt (Ser473) (p-Akt), Akt, phospho-p70S6 kinase (Thr421/Ser424) (p-p70S6K), p70S6K, eukaryotic initiation factor 4E-binding protein-1 (4E-BP1), HIF-1α, Notch-1, c-Jun N-terminal kinases (JNK), p38 and phosphor-Forkhead Box O1 (Thr24) (p-FoxO1) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Antibodies against phosphor-mammalian target of rapamycin (p-mTOR) (Ser2448), mTOR were purchased from R&D Systems (Minneapolis, MN, USA). Antibodies against phosphor-4E-BP1 (Ser65/Thr70) (p-4E-BP1), c-Myc, phosphor-extracellular signal-regulated kinases 1/2 (Thr202/Tyr204) (p-ERK1/2), ERK1/2 and GAPDH were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz). Plasmids (myrAkt delta4-129, pcDNA3-Flag mTOR wt, pWZL Neo Myr Flag RPS6KB1, pET14b PHAS-I, HA-HIF1alpha-pcDNA3, 3XFlagNICD1, pMXs-hc-Myc, ODD-Luciferase-pcDNA3, VEGF promoter, cMyc promoter (TBE1/2-wt), and pCBFRE-luc) were purchased from Addgene (Cambridge, MA, USA).
9
Inhibiting Notch Signaling in Colorectal Cancer
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Oxaliplatin and 5FU were purchased from the Colorectal Cancer Institute of Harbin Medical University. The γ-secretase inhibitor N-[N-(3,5-difluoroph enacetyl)-l -alanyl]-S-phenylglycine t-butyl ester (DAPT) was purchased from Sigma-Aldrich and was used to inhibit Notch signaling in vitro and in vivo. The antibodies used for flow cytometry, immunohistochemistry (IHC), immunofluorescence and western blot analysis were as follows: Rabbit anti-cluster of differentiation (CD) 133, rabbit anti-Notch1 (Cell Signaling Technology, Inc., Beverly, MA, USA), rabbit anti-β-actin (Sigma-Aldrich), mouse anti-CD44 (Abcam PLC, Cambridge, UK), allophycocyanin (APC)-conjugated anti-CD133, APC-conjugated mouse-immunoglobulin G (IgG) 1 (Miltenyi Biotec, Bergisch Gladbach, Germany), phycoerythrin (PE)-conjugated anti-CD44, PE-conjugated mouse-IgG2b (Becton, Dickinson and Company, Franklin Lakes, NJ, USA), rabbit anti-hairy and enhancer of split-1 (HES-1; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and mouse anti-Ki67 (Dako, Ely, UK).
10
Pharmacological treatments in zebrafish
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All pharmacological treatments were performed as described previously (López-Schier and Hudspeth, 2006 (link); Wibowo et al., 2011 (link); Pinto-Teixeira et al., 2015 (link)). Briefly, the following concentrations and timings used were: Neomycin sulfate (Sigma, St. Louis, MO) 250 µM for 45 min; N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine-t-butyl ester (DAPT) (Sigma) 100 µM for 24–48 hr. Equal amounts of DMSO were diluted in embryo medium for control specimens.
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