Figure 5. TTBK2 localizes to the basal body in response to serum withdrawal.

(A-D) Localization of TTBK2 fusion protein constructs expressed in mouse cells. (A, B) WT MEFs were infected with a mouse N-terminal-eGFP-TTBK2 construct. TTBK2-eGFP (green) is present in the transition zone between the mother centriole, labeled with γ-tubulin (purple), and the axoneme, labeled by acetylated α-tubulin (red), in ciliated cells (A), and is localized to one of the two centrosomes in non-ciliated cells (B). (C, D) WT MEFs infected with mouse TTBK2 with a C-terminal V5 tag (green); TTBK2-V5 also localizes to the transition zone (C). TTBK2-V5 co-localizes with IFT88 (red) in the transition zone (D). Insets show high magnification images that highlight the overlap of IFT88 and TTBK2 at the base of the cilium. (E) An antibody to human TTBK2 (green) shows that the endogenous protein localizes to the transition zone of human RPE cells. Scale bar = 2 μm. (F-K) WT MEFs stably expressing mTTBK2::eGFP after shift to serum-free medium. 48 hours of serum withdrawal, cells were shifted back to growth media induce re-entry into the cell cycle (K). Localization of mTTBK2::eGFP was assessed using an antibody against GFP (green), and cells were counterstained with γ-tubulin (purple) to label the centrosome and acetylated α-tubulin (red) to label the ciliary axoneme. (L) Line graph shows the mean percentage of cells with centrosomal localization of mTTBK2::eGFP (green) the mean percentage of ciliated cells (red), and the mean percentage of cells with CP110 on both centrioles (black) at each time point from 4-6 randomly selected fields of cells from at least two different slides; see Supplemental Methods. Error bars indicate SEM. See also Figure S5.