Figure 3: ILEI knockdown selectively stabilizes APP-CTFs.

(a) Immunoblots for APP and its proteolysed derivatives in control and ILEI-knockdown (k/d) HEK293 cells. The same amount of protein from cell lysates (for ILEI, APP FL and APP-CTFs) or conditioned medium (for secreted ectodomain; sAPP) was loaded. For APP-CTFβ, a longer exposure image is shown. m, mature; im, immature. (b) Immunoblots of APP-FL and APP-CTF in a cycloheximide (CHX) chase assay. m, mature; im, immature. Non-targeting control or ILEI-specific siRNA-transfected HEK293 cells were treated with 50 μg ml⁻¹ CHX for the indicated times. β-Actin was used as a loading control. The graphs below show the relative intensity of the bands (n=5, mean±s.d.). **P<0.01 versus the control by Student’s t-test. (c) The restoring effect of siRNA-resistant ILEI-V5-His (srILEI-VH) on the levels of APP-CTFs and secreted Aβ. ILEI-specific siRNA-transfected HEK293 cells were further transfected with srILEI-VH. APP-CTF was analysed with immunoblotting, and secreted Aβ40 was measured with ELISA (n=3, mean±s.d.). **P<0.01 by Student’s t-test. (d) Immunoblots for LRP1-CTF (25 kDa), N-cadherin-CTF (36 kDa) and APP-CTF (10 kDa) in control, stable ILEI-knockdown (k/d), stable ILEI-VH-overexpressing (oe) and DAPT (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester; 1 μM)-treated HEK293 cells. β-Actin was used as a loading control.