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. 2000 Sep;11(9):3073–3087. doi: 10.1091/mbc.11.9.3073

Figure 6.

Figure 6

Biochemical requirements for BFA-induced Golgi disassembly (A) and Golgi tubule fusion (B) in semi-intact cells. (A) Semi-intact CHO-GT cells were incubated with either BFA/ATP/cytosol (cyto), BFA/ATP/NEM-treated cytosol (NEM-cyto), or BFA/ATP/NEM-treated cytosol/DTT (NEM-cyto+DTT) at 32°C for 80 min, fixed, and then subjected to morphometric analysis (Golgi disassembly assay). The protein concentration of the cytosol used in this experiment was 1.7 mg/ml. (B) At first, semi-intact cells were incubated with BFA/ATP at 32°C for 80 min to generate the Golgi tubules. About 65% of cells had Golgi tubules. Then the cells were further incubated with BFA/ATP/NEM-treated cytosol (NEM-cyto), BFA/ATP/NEM-treated cytosol/myc-NSF (myc-NSF), BFA/ATP/NEM-treated cytosol/His-NSF (His-NSF), or BFA/ATP/NEM–treated cytosol/K266Q-NSF (inactive NSF) at 32°C for 80 min, and morphometric analysis was performed (Golgi tubule fusion assay). The protein concentration of the cytosol used was 2.3 mg/ml. Three hundred cells were counted for each sample. Cells were counted in three randomly selected fields. Three independent experiments were performed, and means and SDs were calculated.