Figure 5: The localization and function of DYF-19 are highly conserved.

Either endogenous (a) or overexpressed (b) FBF1, the mammalian homologue of worm DYF-19, localizes specifically on one centriole with a ring-like pattern in IMCD3 cells. (c) FBF1 localizes at the ciliary base, above the basal body. (d) In IMCD3 cells, FBF1 localizes above rootlet and sub-DAP ODF2 and completely co-localizes with DAP CEP164. (e) Immuno-electron microscopy demonstrates that FBF1 localizes specifically to distal appendages of mother centrioles. (f–h) Knockdown of FBF1 leads to severely truncated cilia in most RNA interference (RNAi)-treated hTERT-RPE cells. Data are represented as mean of three independent experiments (n=200) and error bars indicate s.d. Significant differences were identified by the Student’s t-test. *P<0.001. (i,j) The IFT-B component IFT88, but not the IFT-A component IFT140, enters the truncated cilia of FBF1 knockdown hTERT-RPE cells. Arrows indicate the tips of truncated cilia. (k) Endogenous IFT54 immunoprecipitates with FBF1 in HEK293 cells. (l) HEK293 cells were transiently transfected with FLAG-HA-tagged FBF1, and 48 h later cells were subjected to immunoprecipitation using normal mouse IgG (mIgG) or anti-IFT54 antibody. Fifty micrograms of protein were loaded into each lane. Scale bars, (d) 1 μm; (e) 200 nm; others, 20 μm.