Fig. 3: Identification of ENSA as a substrate for NEP.

A Immunoblotting of ENSA incubated with or without NEP and mentioned inhibitors for 24 h at 37 ˚C. Thio: Thiorphan, Phos: Phosphoramidon. B Specific peak of full-length of ENSA after incubation with or without NEP and thiorphan. C Specific peak of cleaved ENSA after incubation with or without NEP and thiorphan. D Sequence of full-length of ENSA. Arrowheads indicate cleavage site by NEP. E, F Immunoblotting of ENSA from cortices and hippocampi of 6-month-old WT and Mme KO mice. Values indicated in the graph show ENSA band intensities normalized to that of β-actin (n = 5 for each group). G–I Immunoblotting of (H) NEP and (I) ENSA from hippocampi of 3-month-old WT mice after overexpression of active or inactive mutant NEP by SFV gene expression system. Values indicated in the graph show NEP and ENSA band intensities normalized to that of β-actin (n = 4 for each group). J Aβ₄₂ ELISA of Tris-HCl-buffered saline-soluble fractions containing 1% Triton-X from hippocampi of WT mice after overexpression of active or inactive mutant NEP by the SFV gene expression system (n = 4 for each group). K Immunostaining of ENSA (Green), NEP (Red) and DAPI (Blue) in CA3 from 3-month-old WT, Ensa KO and Mme KO mice. Scale bar is 50 µm in low-magnification image and 10 µm in high-magnification image. White arrows indicate colocalized signals. Data represent the mean ± SEM. *P < 0.05, ****P < 0.0001 (Student’s or Welch’s t test).