Figure 4.
Localization of Nek8 to centrosomes and cilia is regulated by the RCC1 domain. (A) hTERT-RPE1 cells were either mock transfected or transiently transfected for 24 h with GFP-Nek8-truncated constructs, as indicated. They were fixed in methanol and stained with antibodies against GFP to detect the recombinant Nek8 (green in merge) and γ-tubulin to detect the centrosome (red in merge). (B) The histogram shows the % cells with the GFP-Nek8 protein visible at the centrosome. (C) hTERT-RPE1 cells were transiently transfected for 4 h with GFP-Nek8 constructs, as indicated, and then serum starved for 48 h to promote ciliogenesis before methanol fixation and staining with antibodies against GFP to detect the recombinant Nek8 (green in merge) and acetylated tubulin to visualize the cilia (red in merge). (D) The histogram shows the % cells with the GFP-Nek8 protein visible at the primary cilium. In (A) and (C), DNA was stained with Hoechst 33258 (blue in merge); insets show enlargements of the cilia; scale bars, 10 µm. In (B) and (D), data represent mean (±SD) of three separate experiments where at least 45 cells were counted.