Figure 4: Secreted ILEI interacts with the PS1/γ-secretase complex and interferes with its APP-CTF-stabilizing property. | Nature Communications

Figure 4: Secreted ILEI interacts with the PS1/γ-secretase complex and interferes with its APP-CTF-stabilizing property.

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

Figure 4

(a) Effects of purified ILEI-VH (pILEI-VH) on Aβ secretion and cellular APP-CTFs. pILEI-VH was added to the culture medium of ILEI-knockdown HEK293 cells at the indicated concentrations (μg ml−1). V5-His tag peptide (VH, 7 μg ml−1) served as a negative control. Secreted Aβ40 was measured with ELISA assay and APP-CTFs were analysed with immunoblotting. The right graph shows the relative intensity of APP-CTFs normalized to β-actin (n=3, mean±s.d.). (b) Laser scanning confocal microscopic images of pILEI-VH. pILEI-VH was immunostained with anti-V5 tag antibody (green) 0 or 4 h after its addition to the medium. Nuclei were stained with Hoechst 33342 (blue). Scale bar, 10 μm. (c) Co-immunoprecipitation assays for pILEI-VH and PS1. ILEI-knockdown HEK293 cells were lysed 0 or 4 h after addition of pILEI-VH, and the cell lysates were subjected to immunoprecipitation. A blot of the anti-ILEI precipitate was probed with anti-PS1-CTF or anti-V5 antibody. (d) ILEI-knockdown effects on APP-CTFs and secreted Aβ40 levels in PS1/PS2-knockdown, PEN-2-knockdown, or DAPT (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester; 1 μM)-treated HEK293 cells (n=3, mean±s.d.). (e) Co-immunoprecipitation assays for PS1 and APP-CTFs. Cell lysates of native, ILEI-knockdown (k/d) and ILEI-overexpressing (oe) HEK293 cells were immunoprecipitated with anti-PS1 antibody (PS1-IP). (f) Co-immunoprecipitation assays for PS1 and ILEI. Cell lysates of native and Swedish APP-overexpressing (Sw-APP) HEK293 cells were immunoprecipitated with anti-PS1 antibody (PS1-IP). (g) Effects of PS1 mutants on ILEI function. Mock, D385A-PS1, CTm2-PS1 or TMD2m-PS1 was transfected into native, ILEI-knockdown (k/d) and ILEI-overexpressing (oe) HEK293 cells (the same cell lines as in e). The same amount of protein from cell membrane lysates was subjected to immunoblotting for APP-CTFs. α: APP-CTFα, β: APP-CTFβ. The graph shows the relative intensity of APP-CTFs normalized to native HEK293 cells transfected with the same plasmid (n=3, mean±s.d.). For data from a, d and g, two-tailed Student’s t-test was used to analyse statistical significance. *P<0.05, **P<0.01 and NS, not significant, versus control (a,d) or native (g).

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