Selection of PF11, a recombinant antibody specific for palmitoylated PSD-95. (A) PSD-95-GFP-PSTCD metabolically labeled with [³H]palmitic acid in HEK293T cells was analyzed by fluorography ([³H]palm) and Coomassie brilliant blue (CBB) staining. Closed and open arrowheads indicate the positions of palmitoylated and nonpalmitoylated PSD-95, respectively. (B) Palmitoylated PSD-95-GFP-PSTCD was purified to near homogeneity. (C) A combinatorial recombinant antibody library was screened in vitro against palmitoylated PSD-95. A promising clone, PF11, was obtained. (D) Indirect PF11 immunofluorescence. The signal (hPF11, red) disappeared in PSD-95 knockdown neurons (shPSD-95, green, arrows). Bars, 20 µm (5 µm, magnified). (E) hPF11 (green) and PSD-95 (red) antibodies showed the similar staining pattern in the hippocampus (top) and cerebellum (bottom) of adult mouse brain sections. Glutamatergic presynapses were labeled with the mixture of vGlut1 and vGlut2 antibodies (blue). DG, dentate gyrus; Mo, molecular layer; PC, Purkinje cell layer; Gr, granule cell layer. Regions in the molecular layer of DG and cerebellum are magnified (right panels). Bars: (top) 500 µm; (bottom left) 100 µm; (bottom right) 2.5 µm. (F) HEK293T cells were cotransfected with DHHC2 and either PSD-95 wild-type (WT) or cysteine-mutated palmitoylation-deficient PSD-95 CS, and cells were stained with hPF11 (red). Bar, 10 µm. (G) hPF11 staining (red) was detected in neurons expressing shPSD-95 (marked by GFP, blue pseudocolor) and complemented by shRNA-resistant (res) PSD-95 WT (stained by anti-PSD-95, green pseudocolor), but not by palmitoylation-deficient resPSD-95 CS. Bars, 10 µm (1 µm, dendritic spines magnified).