NEP enzymatic activity assay

NEP enzymatic activity assay was performed with the Neprilysin Activity Assay Kit (Fluorometric) (BioVision, Milpitas, CA, USA). Cells or brain tissues were lysed in ice-cold NEP Assay Buffer containing 1 mM PMSF (Beyotime) and 10 μg/mL Aprotinin (Sigma, A1153). Protein concentrations were measured by using the BCA Protein Assay Kit (Beyotime, China). Then, 10 μg of total protein was placed in the wells of a 96-well white opaque plate before adding the substrate working solution. Reagents were mixed by shaking the plate gently for 30 s, and the fluorescence signal was immediately measured at Ex/Em = 330/340 nm on the kinetic mode at 37 ºC. The data were recorded every 5 min for 1 h. The NEP-specific activity is expressed as U/mg of protein.