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. 2009 Feb 27;4(2):e4647. doi: 10.1371/journal.pone.0004647

Figure 8. Down regulation of A-RAF by either AR149 or A-RAF siRNA interferes with activation of ARF6.

Figure 8

A. Interaction of AR149 and ARF6. COS7 cells were co-transfected with GFP-AR149 and either HA-tagged ARF6wt, GTP-locked [ARF6(Q67L)] or GDP-locked [ARF6(T27N)]. After immunoprecipitation with α-GFP antibodies, co-precipitated ARF proteins were detected with α-HA antibodies. In the second experiment, precipitation and detection antibodies were exchanged. Expression levels in whole cell lysates (WCL) is shown in two bottom panels. Empty vectors were used for control (bottom panel). B. GGA3 interacts with ARF6•GTP. COS7 cells were transfected with wild type, GTP-locked (Q67L) or GDP-locked (T27N) HA-ARF6. Proteins pulled-down by incubation with GST-GGA3-Sepharose were detected with α-HA antibodies. C.,E. AR149 suppresses EGF-stimulated ARF6 activation. COS7 cells, transfected with either ARF6 alone or ARF6+GFP-AR149, were treated with EGF for 10 min and subjected to GGA3 pull-down. Bound protein was analysed by immunoblotting. Note decrease in the amount of ARF•GTP by AR149 coexpression. AR149 remains in the pulled-down ARF6 complex confirming the immunoprecipitation data. D. AR149 inhibits ARF6 activation by EFA6. COS7 cells were transfected with HA-ARF6 and either AR149, EFA6, or both. ARF6•GTP was pulled-down by GST-GGA3. AR149 decreases EFA6-stimulated ARF6 activation. F. ARF6 activation by EGF or EFA6 requires A-RAF. ARF6 activation was assessed by GGA3 pull-down. The amount of A-RAF and ARF6 protein were determed by Western blotting (WB). Treatment conditions are as indicated.