Figure 3.
Effect of cytD and nocodazole on the internalization of basolateral membrane-inserted C6-NBD-SM and C6-NBD-GlcCer. HepG2 cells were preincubated with or without 10 μg/ml cytD or 33 μM nocodazole in HBSS at 37°C for 30 min or 2 h, respectively. Cells were then cooled and, in the presence of the drugs, labeled with 3 μM C6-NBD-SM or C6-NBD-GlcCer at 4°C. Subsequent incubations were carried out at 37°C for the indicated time intervals. After the incubation, the cells were washed with cold PBS, back-exchanged, and scraped from the culture dish. Lipids were extracted and NBD-lipids were quantified as described in MATERIALS AND METHODS. Each value represents the mean of two independent experiments with variations between 3 and 14%. (A) Internalization of C6-NBD-GlcCer in control (○), cytD (□), and nocodazole-treated (▵) cells. (B) Internalization of C6-NBD-SM in control (•), cytD (▪), and nocodazole-treated (▴) cells.