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Fig. 10. JFC1 binds specifically to Rab8-GTP. (A) Table showing interactions of JFC1 in the two-hybrid system. Positive interaction (+) is indicated by growth in the absence of leucine and by enhanced ß-galactosidase activity using XGal as substrate. (B) Hela cells were transfected by a myc-JFC1 encoding construct, and then the cells were incubated with cytochalasin D for 30 minutes, and stained by anti-myc and anti-Rab8. Endogenous Rab8 colocalizes with myc-JFC1 on tubular structues. (C) In vitro translated Rab2, Rab8, Rab27a and laminin proteins were incubated with GST-JFC1 beads, and bound proteins were analyzed by SDS-PAGE. (D) Cell lysates from cells transfected with contructs encoding GFP-Rab8-Q67L, GFP-Rab8-T22N and mock control were incubated with GST-JFC. Bound proteins were eluted and analyzed by western blotting using anti-Rab8. Note that GST-JFC1 binds GFP-Rab8-Q67L and endogenous Rab8, but not GFP-Rab8-T22N. (E) In vivo binding of JFC1 to Rab8. The construct encoding myc-JFC1 was co-transfected with constructs encoding GST-Rab8-Q67L, GST-Rab8-T22N or GST. Cell lysates from these cells were bound to glutathione-Sepharose beads, and bound proteins were analyzed by westerm blotting with anti-JFC1 and anti-GST antibodies. Only GST-Rab8-Q67L bound JFC1 in vivo. Bar, 10 µM.
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