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Fig. 2. (A) COS-7 cells were co-transfected with recombinant, full-length kinesin II tagged with a myc epitope and either recombinant HA-tagged GSSF1 (a,b) or GSSF5 (c). Immune complexes were precipitated with an anti-HA antibody (a,c) or a control IgG (b) and western blots were stained with anti-KIF3B. The recombinant kinesin II can be detected in immunoprecipitates from cells expressing GSSF1 (lane a, *) but not GSSF5 (lane c). GSSF5 was used here as a control because it does not bind KIF3B in the two-hybrid system (not shown). Electrophoretic mobilities of molecular weight markers of 110 and 80 kDa are indicated to the left of the figure. (B) Co-localization of GSSF1 and KIF3B. COS-7 cells were transfected with myc-KIF3B (a) or co-transfected with myc-KIF3B and HA-GSSF1 (b,c) or myc-KIF3B and HA-GSSF5 (d). Fixed cells were stained with antibodies to myc-KIF3B (a,b,d) or HA-GSSF1 (c). KIF3B localizes to the Golgi (a and d, arrows) and microtubules (a and d, arrowheads) in cells expressing KIF3B alone (a) and in cells transfected with both KIF3B and the control fragment GSSF5 (d). By contrast, when KIF3B is co-transfected with GSSF1, KIF3B distribution is altered and both proteins co-localize in punctate cytoplasmic structures. (C) DAPI-stained COS-7 cells transfected with C-terminal tail domain of KIF3B. Several examples of bi- and multinucleate cells are shown (a-c).
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