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50 measurements for Aβ40 and Aβ42 were determined using electrochemiluminescent detection of peptides secreted by SH-SY5Y cells stably overexpressing the β-APP C-terminal fragment SPA4CT. All compounds were tested at least twice in independent experiments. Consistent with the profile of γ-secretase modulators, total Aβ peptide levels were constant; selected GSMs were confirmed in a SELDI experiment confirming the appearance of shorter fragments while Aβ42 formation was suppressed. (a) Best, J. D.; Jay, M. T.; Otu, F.; Ma, J.; Nadin, A.; Ellis, S.; Lewis, H. D.; Pattison, C.; Reilly, M.; Harrison, T.; Shearman, M. S.; Williamson, T. L.; Atack, J. R. J. Pharm. Exp. Ther. 2005, 313, 902; (b)
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All compounds were also profiled at least twice in independent experiments to assess their effect on Notch processing: HeLa cells were made to co-express nonfunctional halves of luciferase, one fused to NotchΔE and the other to RBP-Jκ. γ-Secretase mediated cleavage of NotchΔE results in release of Notch intracellular domain (NICD)/N-terminal luciferase, which translocates to the nucleus and binds the RBP-Jκ/C-terminal luciferase to form a functional luciferase enzyme. This split-luciferase complementation system is used to detect NICD levels by measuring total luminescence upon addition of luciferin to lysed cells Typical transition-state analogues (e.g., L-685,458) do not show a window over Aβ 40/42 processing in this assay
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All compounds were also profiled at least twice in independent experiments to assess their effect on Notch processing: HeLa cells were made to co-express nonfunctional halves of luciferase, one fused to NotchΔE and the other to RBP-Jκ. γ-Secretase mediated cleavage of NotchΔE results in release of Notch intracellular domain (NICD)/N-terminal luciferase, which translocates to the nucleus and binds the RBP-Jκ/C-terminal luciferase to form a functional luciferase enzyme. This split-luciferase complementation system is used to detect NICD levels by measuring total luminescence upon addition of luciferin to lysed cells. Paulmurugan, R.; Gambhir, S. S. Anal. Chem. 2005, 77, 1295. Typical transition-state analogues (e.g., L-685,458) do not show a window over Aβ 40/42 processing in this assay.
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