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Figure 6


Fig. 6. Biochemical analysis of IFT57 mutants. (A) Western blot of lysates generated from the larval heads of 4 dpf wild type and IFT57 mutants. Mutation of IFT57 does not significantly alter the abundance of any other IFT protein other than IFT57. (B) Lysates of wild type (+/+) or IFT57 mutants (IFT57–/–) were incubated with rabbit antibodies against zebrafish IFT88 or normal rabbit IGG as a negative control. Immunoprecipitates were subsequently blotted with antibodies against KIF3A, IFT52 and IFT20. In IFT57 mutants, IFT20 is not precipitated along with the IFT complex, whereas more KIF3A is precipitated. (C) The addition of 1 mM ATP to lysates blocks the association of KIF3A with the IFT particle in wild-type lysates but not in IFT57 lysates. Lysates were prepared as described above, and then ATP was added to a final concentration of 1 mM prior to the addition of IFT88 antibody and precipitation. (D) The addition of 10 mM ATP blocks the association of KIF3A with the IFT particle in both wild-type and mutant lysates. Asterisks indicate IGG heavy chain band from precipitating antibody on immunoblots.





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