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Figure 7


Fig. 7. BFA treatment induces a preferential association of adaptor complexes with both soluble vimentin subunits and insoluble vimentin networks. (a) Immunoprecipitations were performed from rat brain cytosol (lanes 1, 2, 4 and 5) or in the absence of cytosol (lane 3) using either beads coated with no antibody (lane 1), control pre-immune antibodies (lane 2) or polyclonal {sigma}3 (AP-3) antibodies (lanes 3-5). Immunoprecipitated proteins were resolved by SDS-PAGE and transferred to membranes that were incubated with (lanes 1-4) or without (lane 5) recombinant vimentin (Overlay: Vimentin). A band of 120 kDa specifically binds vimentin and co-migrates with the ß3 AP-3 subunit, shown by immunoblotting using an antibody directed against ß3B (IB: AP-3). Inputs correspond to 10% of the cytosol used for immunoprecipitation. (b) SW13 v+ cells stably expressing GFP-vimentin were treated with 10 µg/ml BFA for 30 minutes at 37°C and were processed for immunofluorescence. Cells were stained with monoclonal antibodies to the {delta} subunit of AP-3. Confocal microscopy of staining revealed that BFA treatment induced bundling of intermediate filament networks and that bundles were decorated with AP-3 immunoreactivity. Bar, 10 µm. (c) SW13 vimentin positive (v+) and negative (v-) cells were either treated or not with 10 µg/ml BFA for 30 minutes at 37°C. Cells were then extracted with 1% Triton X-100 at 4°C. Detergent-insoluble extracts (lanes 1 and 2) and input (lanes 3 and 4) were analyzed by immunoblot with AP-3 antibodies ({delta} and {sigma}3, the latter not shown), AP-1 antibodies (against the {gamma} subunit), vimentin and tubulin antibodies. Association of both AP-3 and AP-1 with insoluble vimentin networks is increased by BFA treatment. (d) Quantification of the results presented in c. Data is presented as mean fold increase (± s.e.m.) over the amount of adaptor present in the detergent insoluble pool of SW13 v- cells (lane 1) normalized to tubulin (n=3). (e) Soluble extracts from SW13 v- and v+ cells treated or not with 10 µg/ml BFA were immunoprecipitated using antibodies to AP-1 ({gamma}) or AP-3 ({delta}). Immunoprecipitations were then analyzed by immunoblot for the presence of vimentin. BFA treatment induced an association of soluble vimentin protein with both AP-1 and AP-3 adaptors.





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