Figure 2: ILEI inhibits cellular γ-secretase cleavage of APP but not Notch. | Nature Communications

Figure 2: ILEI inhibits cellular γ-secretase cleavage of APP but not Notch.

From: The FAM3 superfamily member ILEI ameliorates Alzheimer’s disease-like pathology by destabilizing the penultimate amyloid-β precursor

Figure 2

Non-targeting control or ILEI-specific siRNA (a), or mock or ILEI cDNA (b) was transiently transfected into HEK293 cells. Aβ40 (black bars) and Aβ42 (grey bars) in the conditioned medium were measured using ELISA assays (n=6, mean±s.d.). **P<0.01 versus the control or the mock by Student’s t-test. Lower panels show immunoblots of ILEI. (c) Immunoblot for APP intracellular domain (AICD) in control, ILEI-knockdown (k/d) or ILEI-overexpressing (oe) HEK293 cells. Cells were treated with 200 μM N-ethylmaleimide (NEM) for 1 h to block degradation of AICD60. AICD production was inhibited by DAPT (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester; 1 μM) treatment (d). The graph shows the relative intensity of AICD normalized to β-actin (n=3, mean±s.d.). **P<0.01 versus the control by Student’s t-test. (d) Proteolysed derivatives of endogenous Notch-1 in control and ILEI-knockdown (k/d) mouse embryonic fibroblasts (MEFs). EDTA (1.5 mM) treatment induced cleavage of transmembrane/intracellular Notch (TMIC) to generate Notch extracellular truncation (NEXT), which was then cleaved by γ-secretase to release Notch intracellular domain (NICD). NICD production was inhibited by DAPT (1 μM) treatment. The graph shows the relative intensity of NICD normalized to β-actin (n=3, mean±s.d.). No significant difference versus the control was found (P>0.05 by Student’s t-test).

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