Figure 5.

BFA-stimulated loss of lipid droplets is blocked by inhibitors of mono-ADP ribosylation. (A) CHO K2 cells were incubated in the presence of 5 μCi of [³H]oleate for 24 h to label neutral lipids, washed, and then incubated further in the presence of 2 μg/ml BFA or EtOH carrier for 12 h at 37°C in the presence or absence of 30 mM NAM. Droplet-enriched fractions were prepared, and the lipid was extracted with acetone and counted. Each bar is the average of three experiments ± SE. (B) CHO K2 cells were incubated in the presence of 2 μg/ml BFA or EtOH carrier plus or minus 30 mM NAM. Purified droplets were prepared, and equal-volume fractions were separated by 12% SDS-PAGE and immunoblotted with α-ADRP and α-Rab18 IgG. (C) NRK cells were processed as described in Figure 1B to increase the number of droplets before incubating the cells in the presence of 2 μg/ml BFA plus 30 mM NAM for the indicated time. Cells were stained with α-ADRP IgG and oil red O. Bar, 10 μm. (D) The experimental design is the same as described in A, except that 100 μM MIBG was used instead of NAM. Each bar is the average of triplicate measurements ± SE.