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. 2018 Feb 5;217(2):715–730. doi: 10.1083/jcb.201705184

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© 2018 Wigerius et al.

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Figure 3. — AMOT-130 knockdown destabilizes PSD scaffolds and triggers spine elongation. (A) Rat IEC-18 lysate transfected with control (CsiRNA) or AMOT-130 siRNA (si130#1 and si130#2) was analyzed by immunoblotting with the indicated antibodies. Numbers indicate residual amounts of AMOT-130 relative to control. (B) Hippocampal neurons transfected with siRNA together with GFP-actin or with RNAi resistant (RNAiR) AMOT-130 constructs were immunostained for endogenous PSD-95 and Homer. Bars, 5 µm. (C) Quantification of dendritic spine length in neurons transfected with siRNA. (D) Cumulative frequency plot of spine lengths. (E) Quantification of dendritic spine density. P = 0.55. (F) Characterization of spine shapes classified as filopodia/thin, mushroom, or stubby spines on neurons transfected with siRNA. (G) Quantification of PSD-95 and Homer cluster intensity in AMOT-130–depleted neurons. Error bars represent means ± SD. *, P < 0.05; **, P < 0.01; one-way ANOVA followed by Tukey’s post hoc test. (H) CsiRNA or si130#1 electroporated into dissociated hippocampal neurons at 0 DIV before plating. Lysates were analyzed by immunoblotting with AMOT antibody and reprobed against PSD-95, Homer, and tubulin. Molecular masses are given in kilodaltons.