Figure 7.

Cby facilitates recruitment of Rab8 to the basal bodies via stabilization of the CEP164–Rabin8 complex. (A) MTECs were fixed at ALId4 and immunostained for γ-tubulin and Rab8. Top-down (x-y) and side (y-z on the right and x-z on the top or bottom) views are shown. Bar, 5 µm. (B) Quantification of the number of ciliated cells with apical Rab8 at the indicated ciliogenesis stages. MTECs were fixed at ALId3 through d6 and colabeled with γ-tubulin and Rab8 antibodies. For each genotype, 51–108 ciliated cells were counted per MTEC preparation, and data are presented as means ± SEM from four independent MTEC preparations. *, P < 0.05; ***, P < 0.001. (C) Cell lysates were prepared from WT or CbyKO MTECs at ALId0, d5, and d14 and subjected to Western blotting for Rab8, Rabin8, Cby, and GAPDH as indicated. The asterisk indicates a nonspecific band. The band intensities of Rab8 and Rabin8 were quantified and normalized to those of GAPDH. The normalized values for WT samples at ALId0 were set as 1. (D) HEK293T cells were transfected with GFP or GFP-Rabin8 and Flag-Cby expression plasmids as shown, and cell lysates were immunoprecipitated with the Flag antibody and probed with the GFP antibody. (E) Cell lysates were prepared from HEK293T cells expressing HA–CEP164-CC, Flag-Rabin8, and increasing amounts of untagged Cby, which was immunoprecipitated with the Flag antibody and detected with the HA, Cby, or Flag antibody as shown. IB, immunoblot; IP, immunoprecipitation.