pcDNA3-flag-hAPP695 was as previously described [50 (link)]. pcDNA4/V5-His-hAICD59 expressing the C-terminal 59 amino acid was subcloned from full-length hAPP695. SH-SY5Y cells were obtained from ATCC and grown in Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) FBS. SH-SY5Y cells were transfected with various plasmids using the Lipofectamine 2000 reagent (Invitrogen) according to the instructions of the manufacturer. Two days after transfection, the cells were used for experiments. The SH-SY5Y cell line-overexpressing APP (SH-SY5Y-APP) was generated by transfection with a pcDNA4 plasmid construct (Invitrogen) carrying APP695 and a Zeocin resistance gene followed by selection for Zeocin resistance. To inhibit AICD degradation, the cells were treated with 10 mM NH4Cl overnight in the culture medium [57 (link)]. L-685,458 (5 μM final concentration; Tocris, catalog no. 2627) or BMS 299897 (1 μM final concentration; Tocris, catalog no. 2870) were administered to the cells in culture medium for 24 h.
L 685 458
Manufactured by Bio-Techne
L-685,458 is a laboratory research chemical produced by Bio-Techne. It functions as a selective γ-secretase inhibitor. Detailed information about its intended use or applications is not available.
Automatically generated - may contain errors
Lab products found in correlation
William s e medium, by Thermo Fisher Scientific (2 mentions)
Interleukin 2 (il 2), by Miltenyi Biotec (2 mentions)
Ifn α, by Merck Group (2 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Bms 299897, by Bio-Techne (1 mentions)
Goat anti human igm igg f ab 2 fragments, by Jackson ImmunoResearch (1 mentions)
Cytation 3 plate reader, by Agilent Technologies (1 mentions)
8 protocols using l 685 458
1
Characterizing APP and AICD Regulation
2
Inhibition of TGFβ and Notch Signaling in 3D Human Cell Culture
3
Comprehensive B-cell Activation Protocol
4
Exploring Jagged1 and Notch1 in Liver dECM
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Samples of liver dECM alone, 2D cultured SV40SM, and dECM-encapsulated SV40SM were assayed for mouse Jagged1 and Notch1 proteins via ELISA according to manufacturer specifications (LifeSpan Biosciences catalog no. LS-F7394 and LS-F14664-1 respectively). Briefly, samples were harvested after 1 and 7 days of culture and frozen at −80 °C until homogenization on the day of assay. Samples were read on a Cytation 3 Plate Reader (BioTek). Values were normalized to cell number per culture. For Notch inhibition experiments, cultures of encapsulated eGFP-SV40SM were incubated with culture media supplemented with 25 µM of the γ-secretase inhibitor L-685,458 (Tocris) and imaged every 48 hours.
5
Generation and Treatment of HIV-1 with Inhibitors
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WT HIV-1 was generated by transfection of 293T cells with infectious clone pNL4-3 (AIDS Reagent Repository number 114). To generate HIV-1 carrying VSV-G envelope glycoprotein, pNL4-3.Luc.R−.E− plasmid (AIDS Reagent Repository number 3418) was transfected into 293T cells together with a VSV-G-expressing construct (pVSV-G) as described20 (link). γ-secretase inhibitor L-685,458 (Cat # 2627) was purchased from Tocris. β-secretase (BACE1) inhibitor Verubecestat (MK-8931, Cat # S8173) was purchased from Selleckchem. CHME3 4 × 4 or 293T cells were treated with DMSO or γ-secretase inhibitor or BACE1 inhibitor reconstituted in DMSO at 1 μM and/or 2.5 μM 4 h or 6 h post transfection or infection, respectively, and maintained throughout the entire experiment.
6
Cytokine-Induced B-Cell Activation
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Reagents used included the following: IL-2 (Miltenyi Biotec); IL-21, IL-6, and SDF1 (PeproTech); IFN-α (Sigma-Aldrich); multimeric APRIL H98 and multimeric CD40L (AdipoGen Life Sciences); goat anti-human IgM and IgG F(ab′)2 fragments (Jackson ImmunoResearch); lipid mixture 1, chemically defined (200×) and MEM amino acids solution (50×) (Sigma-Aldrich); and L-685,458 (γ-secretase inhibitor [GSI]) (Tocris).
7
Inhibition of TGFβ and Notch Signaling in 3D Human Cell Culture
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Human CPs were suspended at a density of 8 × 104 cells/ml, in a mixture of 40% matrigel and 60% William’s E medium (Gibco, Life Technologies) with supplements as described above. The cell suspension was distributed in 3 equal volume aliquots. One aliquot received no further supplementation and was used as a positive control. The second aliquot was further supplemented by 10μM SB-431542 for the inhibition of TGFβ/activin signaling. 50μM of L-685,458 (Tocris Biosciences) were added in the third aliquot for the inhibition of Notch signaling. Each aliquot was distributed in 24-well plate format. The same concentrations of inhibitors were added to the medium overlaying the matrigel on a daily basis. After a total of 10 days in 3D culture, the total number of cysts in 4 random wells of a 24 well plate was counted for each condition by a blinded researcher. Error bars represent SD.
8
Drosophila Larvae Reared on Gamma-Secretase Inhibitor
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