(A) RPE1 cells were treated with control siRNA or Nek1 targeting siRNA, serum starved for 48 hr, and stained for acetylated tubulin (red), γ-tubulin (green) and DNA (blue). Scale bar represents 5 μm. (B) siRNA-treated cells were subjected to Western blot for endogenous Nek1 and GAPDH. (C) Cells were transfected with control siRNA, Nek1 siRNA, or Nek1 siRNA together with Cdc20 siRNA respectively, serum starved for 2 days, and stained for acetylated tubulin. The percentage of cells with normal cilia or branched abnormal cilia was calculated from three independent experiments. Data are means ±S.D. (D) Western blot showed the decrease of Nek1 protein levels after serum stimulation. (E) Degradation of 35S-labelled Nek1 was examined in cell extracts prepared from HeLa S3 cells. Emi1 was added to test if the degradation was dependent on APC activity. The data were analyzed by autoradiography. (F) HA-tagged Nek1 was expressed and labeled with 35S in reticulocyte lysate. 35S-labelled Nek1 was purified through HA tag and subjected to in vitro ubiquitylation catalyzed by purified APC. (G) RPE1 cells were transfected with plasmid expressing HA-Nek1 and/or Myc-Cdc20, starved for 2 days and subjected to western blot for Myc tag, HA tag, and GAPDH.