Figure 3: The ankyrin-repeat domain of ANKS6 is necessary for NEK8 binding and targeting of ANKS6 to the IC.

(a) Different ANKS6 truncations were employed in IP/binding assays and IF. (b) ANKS6 interacts with NEK8 through its ANK-repeat domain, while the SAM domain is not essential for binding. ANKS6 truncations that interact with NEK8 are readily phosphorylated in complex with NEK8. (c) A truncation variant of ANKS6 lacking the first ANK repeat binds to NEK8, while truncation of the first four ANK repeats completely abrogates binding. (d,e) Short hairpin RNA (shRNA)-mediated targeted knockdown of NEK8 significantly reduces the ciliary ANKS6 signal. IF signal intensities were compared between wild-type IMCD cells and IMCD^shNek8 cells, for both anti-NEK8 and anti-ANKS6 signals (scale bar, 2 μm). The number of individual cilia analysed is indicated in the respective bar. Differences were highly significant by Student’s t-test, with P<0.01 (upper bar graph). The total level of ciliation is only mildly affected by Nek8 knockdown (lower bar graph; percentage of numeric counts of ciliated cells over total cells). (f) Fluorescence immunohistochemistry on kidney sections of wild-type (upper panels) and Nek8^−/− mice (lower panels), employing antibodies against ANKS6 (green) and acetylated tubulin (red). Positive ANKS6 signals at the ciliary base can only be identified in the wild type, not in Nek8^−/− cilia (scale bar, 5 μm). (g,h) Coexpression of myc-tagged ANKS6 truncation variants and FLAG-tagged wild-type NEK8 demonstrates the importance of the ANK-repeat domain for NEK8 binding and consecutive ciliary targeting of ANKS6 (scale bar, 2 μm; upper bar graph, percentage of numeric counts of ANKS6-positive cilia over total cilia; lower bar graph, percentual level of ciliation; the number of cells counted is indicated in each bar).