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Fig. 5. Kinesin II mediates Rip11/FIP5 binding to microtubules. (A) Glutathione beads coated with GST alone, GST-Kif3A-tail, GST-Kif3B-tail or GST-Kif3C-tail were incubated with HeLa cell Triton X-100 lysates. Beads were then washed and bound Rip11/FIP5 or RCP/FIP1 was analyzed by immunoblotting. The lower molecular size band present only in the GST lane represents a non-specific band since it did not disappear when we use lysates treated with Rip11/FIP5 siRNA (data not shown). (B) Glutathione beads coated with GST alone, GST-Kif3A-tail, GST-Kif3B-tail or GST-Kif3C-tail were incubated with recombinant purified 6His-Rip11/FIP5. Bound 6His-Rip11/FIP5 was detected by immunoblotting. (C) Glutathione beads coated with either GST alone or GST-Kif3B-tail were incubated with Rip11/FIP5(490-652) in the presence or absence of recombinant Rab11a. Bound Rip11/FIP5(490-652) was detected by immunoblotting. (D,E) Rip11/FIP5 binding to microtubules analyzed using microtubule sedimentation assay (see Materials and Methods). Where indicated, assays were performed in the presence off either 5 mM ATP or recombinant Kif3B-tail protein. In the bottom panel, mock-treated or Kif3B siRNA-treated lysates were used in the assay.

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