Figure 5.

Effect of transport inhibitors on palmitoylation of SNAP-25. (A) PC12 cells were labeled with [³H]palmitate or [³⁵S]methionine for 1 h in normal media (control) or in media containing 10 μg/ml BFA, 10 μM monensin, 20 μg/ml nocodazole, nocodazole and BFA together, or 50 mM NH₄Cl. Radiolabeled SNAP-25 was immunoprecipitated from cell lysates. The effect of these drugs on palmitoylation of SNAP-25 was assessed by incorporation of [³H]palmitate and by analysis of the mobility of the [³⁵S]methionine-labeled protein on SDS-PAGE. (B) PC12 cells were pulse labeled for 20 min with [³⁵S]methionine at 37°C and then chased without label for 0.5, 1, or 2 h at 37°C, 20°C, or 15°C. Radiolabeled SNAP-25 was immunoprecipitated and analyzed by SDS-PAGE and fluorography. Note the inhibition of maturation of SNAP-25 at 15 and 20°C.