AMOT-130 is a substrate for Lats1 in hippocampal neurons. (A) Neurons immunostained for Lats1 (green), PSD-95 (red), and MAP2 (blue) were imaged at 18 DIV. Bar, 10 µm. (B) Immunoprecipitation (IP) from 500 µg of E18 or adult rat brain lysate with the indicated antibodies. Resulting immunoblots were probed with Lats1 antibody (top, filled arrowhead) and reprobed with AMOT-130 2095 antibody (bottom, open arrowhead). The asterisk indicates the IgG heavy chain. (C) Analysis of phosphorylated (top) and Lats1 (middle) expression in rat brain at the indicated developmental stages using antibodies as indicated. Quantification of pSer-909 levels normalized to Lats1 protein in E18 and adult samples (bottom). **, P < 0.01; Student’s t test. (D) Neurons (10 DIV) stimulated with vehicle (Veh) or 100 nM Okadaic acid (OA) for 30 min followed by posttreatment with λ-protein phosphatase (λPP) were analyzed with the indicated antibodies (top and middle). Quantification of pSer175 levels normalized to AMOT-130 (bottom). **, P < 0.01; Student’s t test. (E) Neurons were exposed to 100 nM 17-AAG (AG) for 60 min before OA stimulation followed by immunoblotting with the indicated antibodies. Quantification of pSer175 levels normalized to AMOT-130. *, P < 0.05; **, P < 0.01; one-way ANOVA followed by Tukey’s post hoc test. Molecular masses are given in kilodaltons. (F) Immunostaining of Flag (red) in neurons coexpressing Flag-Lats1 together with GFP-actin. (G) Neurons expressing mCherry together with YFP–AMOT-130 and Flag-Lats1. Panels show dendritic sections of mCherry and YFP–AMOT-130 as outlined with punctate clusters along dendritic shafts indicated by arrows. Quantification of YFP–AMOT-130 spine enrichment (Ef). Error bars represent means ± SD. **, P < 0.01; Student’s t test. Bars, 5 µm. (H) Summary of AMOT-130 functions during synapse development (see Discussion).