FIGURE 2.

Regulation of SNAP25/23 membrane binding by DHHC proteins. HEK293T cells were co-transfected with EGFP-tagged versions of SNAP25a, SNAP25b, or SNAP23 together with HA-tagged constructs encoding each of the 23 murine DHHC constructs (or empty HA vector as a control). Twenty-four hours post-transfection, the cells were fractionated into cytosol (C) and membrane (M) fractions, which were resolved by SDS-PAGE and transferred to nitrocellulose for immunoblotting analysis with anti-GFP. The left panel of the figure shows representative immunoblots (top, SNAP25a; middle, SNAP25b; bottom, SNAP23). The numbering corresponds to the specific DHHC protein that was co-transfected with SNAP25/23. The right panel shows averaged data and S.E. (n = 3) for the increase in % membrane association of the SNAP25/23 proteins in the presence of each of the DHHCs. For clarity, the basal level of membrane association in the absence of DHHC co-expression was subtracted for each condition. The data were analyzed using a one-way ANOVA, which revealed significant increases in membrane association with DHHC3/7/17 for each SNAP25/23 protein and for DHHC15 specifically with SNAP25b when compared with transfections in the absence of DHHCs (*, p < 0.05).