Figure 8.

Fractionation of mitotic ER and Golgi after continuous brefeldin A treatment. (A) Subconfluent cultures of HeLa cells were incubated with 0.5 μg/ml nocodazole for 21 h followed by an additional incubation with 10 μg/ml brefeldin A and 0.5 μg/ml nocodazole for 3 h. Mitotic cells were collected by shake-off, and the postnuclear supernatant was fractionated on a 5–25% glycerol velocity gradient. The relative distribution of GPP130 and p63 was determined by immunoblotting. (B) Subconfluent cultures of HeLa cells were incubated with 0.5 μg/ml nocodazole and 10 μg/ml brefeldin A for 21 h followed by complete brefeldin A washout. Cultures were allowed to incubate with 0.5 μg/ml nocodazole for an additional 3 h and analyzed as above.