Fig 1: Identification of ENSA as a NEP regulator in vitro.A-C. NEP activity after treatment of co-cultured cells with 1 µM somatostatin or TT232 for 24 h. A Cortical/hippocampal (Ctx&Hip) neurons (n = 12 wells per treatment), (B) co-cultured neurons (n = 10 wells per treatment), and (C) basal ganglia neurons (n = 8 or 9 wells per treatment) were used. D–F NEP activity in co-cultured neurons after the replacement of the culture medium with conditioned media from (E) Ctx&Hip and (F) basal ganglia neurons treated with 1 µM somatostatin for 0–6 h. n = 6–10 wells per treatment in co-cultured neurons. G–K NEP activity of co-cultured neurons after replacement of the culture medium with separated conditioned media from Ctx&Hip neurons treated with SST or TT232. H, I 10 and (J and K) 30kDa centrifugal filters were used for the separation (n = 7–10 for each group). NEP activity in co-cultured neurons after incubation with (L) ENSA, (M) NSG-1 and (N) NUCKS-1 recombinant proteins for 24 h. n = 8–10 wells per treatment in co-cultured neurons. Data represent the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA with Dunnett’s post-hoc test).