Synaptic distribution of SUMOylated CASK. (A) At 16–17 DIV, cultured hippocampal neurons were divided into three different groups: (1) untreated control, (2) 1 μM TTX overnight, and (3) overnight TTX followed by 60 mM KCl for 3 min. 20–40 min after removing KCl, cells were fixed and stained with SUMO1, CASK, and PSD-95 antibodies. (B) Quantification of SUMO1, CASK, and PSD-95 puncta along dendrites. More than 80 dendrites from >22 neurons were analyzed for each group. Mean values ± SEM (error bars) are shown. ***, P < 0.001 by t test. (C) The percentage of PSD-95, CASK, and PSD-95–CASK double-positive puncta in the population of SUMO1-positive puncta along dendrites. (D) Colocalization of PSD-95 and Bassoon along dendrites of neurons treated with TTX alone and TTX followed by potassium chloride. (E) Quantification analysis of PSD-95 and Bassoon double-positive puncta. More than 80 dendrites from >18 neurons were analyzed in each group. Mean values ± SEM are shown. Bars, 2 μm.