Fig. 3. Arf6(T27N) localizes to detergent-insoluble structures reminiscent of aggresomes. (A,B) When expressed in cells, Arf6(T27N) is essentially insoluble even in the presence of detergent. Cells were transfected with expression plasmids encoding HA-tagged Arf6-wt and the T27N, T44N and Q67L mutants. (A) The cells were homogenized and the post-nuclear supernatants were centrifuged at 100,000 g to give a membrane (m) and a cytosolic (c) fraction. (B) BHK-21 cells were lysed at 4°C in Triton-X-100-containing buffer and centrifuged to separate Triton-X-100-soluble (s) and -insoluble (i) fractions. The different fractions were immunoblotted using anti-HA mouse monoclonal antibody. (C) Expression of Arf6(T27N)-EGFP induces the formation of punctate structures that are regrouped near the nucleus over time in a microtubule-dependent manner. BHK-21 cells were transfected with Arf6(T27N)-EGFP constructs and fixed at different time points. Cells were treated for 2 hours with 30 µM nocodazole before fixation (d). 48 hours after transfection, cells were probed with anti-vimentin antibodies (e-g). At high expression levels, perinuclear Arf6(T27N)-EGFP structures (e) are surrounded with a vimentin ring (f, arrowheads). A merged image is presented (g).