FIGURE 3.

A point mutation in SNAP23 enhances interaction with DHHC15. HEK293T cells were co-transfected with EGFP-tagged SNAP23 or a SNAP23(C79F) mutant together with HA-tagged DHHC15 or empty vector (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 and anti-HA. A, representative immunoblots are shown. B, averaged data and S.E. (n = 4) for the increase in % membrane association of the SNAP23 proteins in the presence of DHHC15 are shown. ** indicates a statistically significant increase in membrane binding (p < 0.01, Student's t test) compared with wild-type SNAP23. C, HEK293T cells were transfected with EGFP-SNAP23 wild-type or C79F mutant together with pEFBOS-HA (control) or HA-DHHC15. Cells were labeled with [³H]palmitic acid, lysed, and resolved by SDS-PAGE. The top panel shows [³H]palmitate incorporation, the middle panel shows expression levels of EGFP-SNAP23, and the bottom panel shows HA-DHHC15 expression. Molecular weight markers are shown on the left of all figure panels.