Essential role of DHHC2-mediated PSD-95 palmitoylation in AMPAR homeostasis. (A and B) Upon 2 µM TTX treatment, TIRFM intensity of pH-GluR1 punctae gradually and continually increased over a 12-h observation. Fluorescence intensity was displayed in pseudocolor and was plotted with time. Kymographs represent the changes in the intensity of pH-GluR1. White lines indicate the regions used for the kymographs. Insets are magnified in the middle panels. (C) Post hoc immunostaining with PSD-95 showed that all GluR1 punctae by TIRFM overlapped synaptic PSD-95 clusters (arrows). (D) Quantification of fluorescent intensities of pH-GluR1 by TIRFM at 12 h after TTX or Kyn treatment. Knockdown of DHHC2 or PSD-95 completely inhibited the homeostatic increase of surface GluR1. n = 3 each; ***, P < 0.001 compared with nontreated control. miLacZ is a control miRNA targeting LacZ. (E) The inhibitory effect of DHHC2 knockdown was rescued by miDHHC2-resistant DHHC2 (WT) but not by PAT-inactive DHHC2 (CS). Furthermore, the inhibitory effect of PSD-95 knockdown was rescued by short hairpin RNA–resistant PSD-95 (WT) but not by palmitoylation-deficient PSD-95 (CS). n = 3 for each; ***, P < 0.001. (D and E) Error bars indicate SD. Bars: (A and B [top] and C) 10 µm; (A and B [insets]) 0.5 µm; (A and B [bottom] and E) 5 µm.