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. 2012 Dec 1;23(23):4543–4551. doi: 10.1091/mbc.E12-05-0336

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© 2012 Ohno et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 3: — The yeast expression system is useful for determining PATs for PSD-95 and SNAP25b. (A, B) SEY6210 (wild type) cells harboring the pYU-PSD95 (PSD-95-9xMyc; A) or pAC-SNAP25b (9xMyc-SNAP25b; B) plasmid were transfected with the pAKNF314 (vector), pAKNF-AKR1 (3xFLAG-AKR1), pAKNF-AKR2 (3xFLAG-AKR2), pAKNF-SWF1 (3xFLAG-SWF1), pAKNF-ERF2 (3xFLAG-ERF2), pAKNF-PFA3 (3xFLAG-PFA3), pAKNF-PFA4 (3xFLAG-PFA4), pAKNF-PFA5 (3xFLAG-PFA5), pAKNF-DHHCx (3xFLAG-DHHCx, with x representing the DHHC number, x = 1–17, 19–22, and 24), or pLECF-DHHC18 (DHHC18-3xFLAG) plasmid. Total cell lysates were prepared and treated sequentially with NEM, hydroxylamine, and biotin-HPDP. (A) The biotinylated samples were subjected to immunoprecipitation with anti-Myc antibodies and detected either by streptavidin-HRP (top) or by immunoblotting with anti-Myc antibody (bottom). (B) The biotinylated samples were precipitated with immobilized avidin beads and detected by immunoblotting with anti-Myc antibody (top). The DHHC proteins in the total lysates were detected using immunoblotting with anti-Myc antibody (bottom). Each experiment was repeated three times, and representative results are shown. pal., palmitoylated; PSD, PSD-95; SNAP, SNAP25b.