PF11 as a conformation-specific intrabody for palmitoylated PSD-95. (A–D) In the presence of DHHC2, PSD-95-mCherry and PF11-GFP were colocalized near the plasma membrane and on intracellular vesicular structures in HEK293T cells (B and C, white arrowheads). Treatment with 2-BP (100 µM, 8 h) dissociated PF11-GFP from the plasma membrane (asterisks) and intracellular aggregates of PSD-95-mCherry (D, black arrowheads). Bars, 10 µm (2 µm, magnified in C). (E) Immunoprecipitation (IP) of PF11-GFP from HEK293T cells. IB, immunoblotting. CC7, unrelated recombinant antibody; closed arrow, PF11-GFP; open arrow, CC7-GFP; closed arrowhead, PSD-95-mCherry. (F) PSD-95 constructs to test the specificity of PF11 binding using methods in Fig. 3, B and/or E. GuK, guanylate kinase domain; S, serine residues replacing palmitoyl cysteines. Red chain, palmitoyl; green, prenyl; blue, myristoyl groups. tag, mCherry or GFP. (G) Blockade of palmitoylation with 2-BP (100 µM, 18 h) dispersed synaptic labeling by PF11-GFP. Red, mCherry as a fill-in marker. Arrows, multiple subspine clusters. Bars, 5 µm (2 µm, magnified). (H) PF11-GFP tracked a synaptic increase of palmitoylated PSD-95 upon TTX treatment. Arrowheads, multicluster spines. Bar, 5 µm. (I) Immunoprecipitation of PF11-GFP from hippocampal neurons treated with 2-BP or TTX. Arrow, PF11-GFP. Closed and open arrowheads are as in Fig. 1 A.