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Fig. 8. Arf6 is linked to the formation of Rab8-tubules. HeLa cells were transfected with constructs expressing Arf6-wt (A,B), Arf6-T27N (C,D) or Arf6-Q67L (E,F). Next day the transfectants were incubated in 0.1 µM cytochalasin D (CD) for 30 minutes at 37°C. The cells were fixed, processed for immunofluorescence and stained to detect endogenous Rab8 and recombinant Arf6. Note the absence of tubules in the Arf6-Q67L expressing cell (arrow) (E,F). (G) Quantification of cells containing Rab8-specific tubular structures after CD-treatment, in the presence of Arf6-wt, Arf6-T22N and Arf6-Q67L were done. A total of about 50 cells were counted per experimental condition. Values, given as percentage of cells (n=50) exhibiting Rab8-specific tubules in each scoring category, from three separate experiments are shown as the mean ±s.e.m. (H) The specificity of the anti-Arf6 antibody was tested by immnuoblotting against mock-transfected and Arf6-Q67L transfected total cell extracts. Bars, 20 µM.
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