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. 2016 Dec 12;113(52):E8482–E8491. doi: 10.1073/pnas.1612963113

Fig. 1.

Fig. 1.

Palmitoylation of PSD95 triggers a change in PSD95 conformation. (A) Schematic of PSD95 (Ch-PSD95-V) and mutated PSD95 (C3,5S-Ch-PSD95-V) FRET sensor constructs. (B) FRET efficiency values for Ch-PSD95-V with changes in palmitoylation. FRET measurements were made in regions of interest in example cells shown in Fig. S3. Data are shown as mean ± SEM (n = 12–30 cells per condition; *P < 0.0001 relative to Ch-PSD95-V). (C) As an additional assay of the changes in PSD95 palmitoylation observed above, we used colocalization with PF11-GFP, a conformation-specific intrabody for palmitoylated PSD95. Representative images of HEK cells expressing PF11-GFP and PSD95 alone (Top Left), with DHHC2 (Bottom Left), with palmostatin B treatment (Top Right), or with palmostatin B treatment and DHHC2 expression (Bottom Right). Increased aggregation and colocalization with PF11-GFP is correlative with an increase in PSD95 palmitoylation. PSD95 alone is not highly palmitoylated (diffuse fluorescence and lack of colocalization with PF11-GFP) compared with its palmitoylation in the presence of DHHC2 and palmostatin B (coaggregation and colocalization with PF11-GFP). (Scale bar: 10 μm.) (D) Click chemistry palmitoylation assay was used to compare the level of Ch-PSD95-V palmitoylation under different conditions. HEK cells transiently expressing Ch-PSD95-V alone or with Myc-DHHC2 and palmostatin B treatment were labeled with 17-octadecynoic acid (17-ODYA). Ch-PSD95-V was immunoprecipitated and treated with biotin-azide under click chemistry reaction conditions, and subsequently analyzed by Western blotting with anti-PSD95 antibody (Top) and streptavidin (Bottom). The space after the first lane is due to cropping of lanes unrelated to the present analysis. PSD95 alone is not highly palmitoylated. In the bar graph, quantification of the normalized band intensities (ratio of streptavidin signal to PSD95 protein) indicates that Ch-PSD95-V palmitoylation increases 30-fold in the presence of DHHC2 and palmostatin B (n = 4 experiments). IP, immunoprecipitation.