Fig. 3.

S-acylation of EGFP-SNAP25B by zDHHC3. HEK293T cells were transfected with EGFP-SNAP25B and pEF-BOS-HA (vector), HA-zDHHC3, or HA-zDHHC3(C157S), or were left untransfected. Cells were then incubated with C14:0, C16:0, or C18:0 fatty acid azides for 4 h at 37 °C. Incorporated fatty acid azides were detected by click chemistry using an alkyne-800 infrared dye. Isolated proteins were resolved by SDS/PAGE and transferred to nitrocellulose membranes. Representative images are shown. (A) Click chemistry signal (Top), anti-GFP immunoblot (Middle), and anti-HA immunoblot (Bottom). Arrowheads denote the position of the EGFP-SNAP25 band (Top and Middle) and HA-zDHHC3 band (Bottom). (B) Following click chemistry, samples were incubated in hydroxylamine (+) or Tris (−) at a final concentration of 1 M overnight before SDS/PAGE. (Top) Click chemistry signal. (Bottom) Anti-GFP immunoblot. The positions of molecular weight markers are shown.