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. 2004 Jan;15(1):99–110. doi: 10.1091/mbc.E03-06-0368

Figure 4.

Figure 4.

BFA causes redistribution of caveolin to LBs; a model system to follow caveolin accumulation in LB. (A–C) Control BHK cells were treated for 2 h with 5 μg/ml BFA. Caveolin distribution was analyzed with the con-cav antibody (red channel) in combination with GM130 (a Golgi marker, green channel) and DAPI (nuclei, blue). Caveolin is detected in LBs (arrows in B). When, after 2 h with BFA, the drug is washed out for 30 min, the recovery is almost complete (arrows C). (D–G) Untreated or OlAc-treated BHK cells were incubated for 2 h with 5 μg/ml BFA and then resolved on sucrose density gradients. (D) In control cells caveolin (rbTL) accumulates in densities corresponding to the PM (fraction 6–7, 20% sucrose; top). Caveolin partially shifts to the top of the gradient (fraction 1) when the cells are preincubated with 50 μg/ml OlAc or treated for 2 h with BFA (middle). The accumulation in the top of the gradient is maximal (6.8%) when cells pretreated with OlAc are incubated with BFA (bottom). Quantification, as described for Figure 2B, was performed from the data in the figure but similar results were obtained in three independent experiments. (E) The LB fraction (1) for each treatment were isolated in parallel and equal volumes resolved by Western blotting. The levels of caveolin after the different treatments (shown in the histograms) was quantified by densitometry and it is indicated as relative to the first lane. (F) Using the same method, we compared the amount of caveolin in fraction 1 corresponding to the gradients from cells treated for different times with BFA, in either 10%SS (left panel) or OlAc-pretreated cells (right panel). In control cells caveolin reaches a peak after 2 h with the drug; however, in OlAc-treated cells high levels of caveolin are sustained after 4 h with BFA. (G) BHK cells were treated for 2 h with BFA and then the drug was washed out for different times. In fraction 1 of control (left panel) or in cells pretreated with OlAc (right panel), the levels of caveolin return to the initial levels after 30 or 45 min, respectively.