The DHHC2/15 subfamily of PSD-95 PATs is regulated by synaptic activity. (A) Activity blockade induces quantitative palmitoylation of PSD-95 but not Gαq. Hydroxylamine (H)-sensitive palmitoylated proteins were purified from treated neurons by the ABE method. The amount of palmitoylated PSD-95 and Gαq was analyzed by Western blotting. T, Tris treatment as a control of hydroxylamine. (B and C) Kyn-induced PSD-95 palmitoylation and synaptic accumulation were reversible upon washing out Kyn. (B) Treatment of hippocampal neurons with Kyn for 2 h enhanced PSD-95 palmitoylation. After washout, PSD-95 palmitoylation level returned to the basal level within 2 h (ABE), with consistent mobility change of PSD-95 (−βME). In contrast, Gαq, GluR2, and GRIP1 palmitoylation did not change upon activity blockade. Kyn-induced palmitoylation changes were quantified. n = 3 each; ***, P < 0.001. Error bars indicate SD. The dashed line (100%) indicates the normalized control level. IB, immunoblot. (A and B) Closed and open arrows indicate the positions of palmitoylated and nonpalmitoylated PSD-95, respectively. (C) The increased accumulation of PSD-95–GFP upon Kyn treatment returned to the basal level at 2 h after Kyn washout. (D) Cultured hippocampal neurons expressing a DN mutant of the DHHC2 and -15 subfamily (DN-DH2/15) were treated with 3 mCi/ml [³H]palmitate for 2 h in the presence or absence of Kyn. Immunoprecipitated PSD-95 was resolved by SDS-PAGE, followed by fluorography ([³H]palm) and Coomassie staining (CBB). Inhibition of glutamate receptor activity with Kyn greatly enhanced PSD-95 palmitoylation. This enhancement was decreased by DN-DH2/15. IP, immunoprecipitation. Bar, 5 µm.