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Fig. 5. In vivo interaction between centaurin-{alpha}1 and KIF13B. (A) Co-immunoprecipitation of FLAG-centaurin-{alpha}1 with GFP-KIF13B. COS cells transfected with the indicated expression vectors were lysed after 2 days and immunoprecipitated (IP) with an anti-GFP antibody. After washing, immunoprecipitates were resolved on SDS-PAGE, blotted onto PVDF membranes and probed with a monoclonal anti-FLAG antibody (IB) to detect FLAG-tagged centaurin-{alpha}1. One-twentieth of the input (cell lysates) was also immunoblotted (IB) with anti-GFP (lower panel) and anti-FLAG (upper panel) antibodies to ensure that GFP-KIF13B and FLAG-centaurin-{alpha}1, respectively, were expressed. (B) Co-immunoprecipitation of endogenous centaurin-{alpha}1 and KIF13B. The lysates of brain were immunoprecipitated (IP) with control pre-immune serum (lane 2) or an anti-centaurin-{alpha}1 antiserum (lane 3) and endogenous KIF13B visualized in the precipitate and cell lysate (lane 1) by immunoblotting with an anti-KIF13B antibody (I). The lysates of brain were also immunoprecipitated (IP) with control pre-immune serum (lane 2) or an anti-KIF13B antiserum (lane 3) and endogeonous centaurin-{alpha}1 detected in the precipitate and cell lysate (lane 1) by immunoblot (IB) using an anti-centaurin-{alpha}1 antibody (II). These experiments were repeated a further two times with similar results.





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