Changes in synaptic activity remodel postsynaptic PSD-95 nanodomains through local DHHC2 activity. (A and B) Neurons were treated with 90 mM KCl for 5 min and recovered for 60 min in the basal medium (wash out, WO). Neurons were stained triply with hPF11 (green), PSD-95 (red), and vGlut1 (blue) antibodies, and the confocal fluorescence intensities of green and red channels at PSD-95–positive clusters were measured. In total, 170–240 clusters from three neurons were analyzed. ***, P < 0.001 by one-way ANOVA with post-hoc Tukey’s test (B). Bar, 5 µm. (C and D) Neurons treated with high K⁺ were analyzed by 2C-STED imaging of PSD-95 (green pseudocolor) and hPF11 (red pseudocolor), and the distance between the peaks with the highest intensity of hPF11 (arrowheads) and PSD-95 (arrows) clusters was measured (as described in Fig. S5 A). The brightness of magnified images with KCl treatment is enhanced. Gray region in D indicates the subresolution range for STED imaging. In total, 150–200 clusters from 10 neurons (two independent experiments) were analyzed. ***, P < 0.001 by Student’s t test (D). Bars, 500 nm (200 nm, magnified). (E and F) Neurons were cotransfected with miR-DHHC2 and HA-resDHHC2-WT or ER (red), and treated as in (A). The confocal fluorescence intensity of hPF11 (green) overlapped with vGlut1 (not depicted) was measured. In total, 230–420 clusters from 8 neurons (three independent experiments) were analyzed. ***, P < 0.001 by one-way ANOVA with post-hoc Tukey’s test. Bar, 5 µm.