Differential subcellular distribution of PSD-95 palmitoylating enzymes. (A) Specificity of antibodies to DHHC2 and -3. The bands detected by anti-DHHC2 (closed arrowheads) and anti-DHHC3 (open arrowhead) antibodies disappeared when protein expression was knocked down by siRNAs. IB, immunoblot; scr, scramble. (B) DHHC2 was enriched in the postsynaptic density (PSD) fractions (Triton X-100–insoluble postsynaptic; closed arrowheads), whereas DHHC3 was detected in only the P3 fraction (open arrowhead). H, homogenate; S, supernatant; P, precipitate; Syn, synaptosome; Sol, Triton X-100 soluble; Ins, Triton X-100–insoluble postsynaptic density fractions. (C) DHHC2 localized in dendrites and the cell body as small vesicular structures, whereas DHHC3 specifically localized at the Golgi apparatus in 18-DIV hippocampal neurons. (D) Effective knockdown of endogenous DHHC2 and -3. Cultured hippocampal neurons were transfected with mCherry-miR RNAi (miDHHC2 and -3) expression vectors at 10 DIV. 18-DIV neurons were stained by DHHC2 or -3 antibody. Note that somatodendritic DHHC2 vesicles and Golgi DHHC3 (arrows) were not stained in mCherry-expressing knocked down neurons (red). Bars: (C [left] and D) 20 µm; (C [right]) 5 µm.