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Fig. 5. In vivo, expression of EFA6 leads to the activation of Arf6-wt but not of Arf6(T44N). (A) BHK-21 cells were transfected with expression plasmids encoding Myc-tagged Arf6-wt, or Arf6(T44N) alone or in combination with a plasmid encoding EGFP-EFA6 and grown in the presence of [32P]-orthophosphate. After immunoprecipitation with the anti-Myc monoclonal antibody, the bound nucleotides were determined by thin-layer chromatography and subjected to autoradiography. The position of GDP and GTP are indicated. (bottom) Quantification of the results from two independent experiments (mean ± SD). (B) BHK-21 cells were transfected with different Arf6 constructs alone or in combination with EGFP-EFA6. Arf6-GTP was isolated by incubation with a GST fusion containing an ARF6 effector domain.





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