Figure 6.

Effect of nocodazole and BFA on MAL-FLAG internalization. (a) COS-7 cells transiently expressing MAL-FLAG were treated with 5 μg/ml BFA for 30 min, followed by sequential incubations in the presence of BFA with anti-FLAG antibodies for 1 h at 4°C and for 30 min at 37°C after removal of the unbound antibodies. (b and c) Transfected cells were incubated with anti-FLAG antibody for 1 h at 4°C. After incubation for 30 min at 37°C to allow the anti-FLAG mAb/MAL-FLAG complex to accumulate in the perinuclear region, cells were incubated for 30 min in the presence of 20 μM nocodazole (b) or 5 μg/ml BFA (c), fixed, permeabilized, and subjected to immunofluorescence analysis with an anti-mouse IgG secondary antibody. Bar, 10 μm.