Cep63 N-terminus is required for centrosomal localisation of Cep63 and Cep152.

<p>(A) Diagram of Cep63 full length and truncation proteins used in the following experiments. Numbers indicate amino acid positions. (B) Cep63 C-terminus interacts with Cep152. Expression of Flag-Cep63 (FL), truncation proteins, or Flag-empty vector control (e) was induced in 293 FlpIn TREX cell lines by incubation with 2 µg/ml doxycycline for 72 hours, then proteins were immunoprecipitated using anti-Flag resin. Western blots show endogenous Cep152, detected by anti-Cep152 (Bethyl), and Cep63 truncations detected by anti-Cep63 (Millipore). Inputs are 5% of the lysate used for Flag IP. Red arrowhead points to Cep63 truncation 1–135 present in the Flag IP. (C-F) U2OS cells were transfected with YFP-Cep63 (FL), truncation proteins, or YFP-empty vector (e) for 48 hours. (C) Cells were stained with anti-Cep152 (red) and γ-tubulin (blue) antibodies; YFP-tagged proteins were detected by direct fluorescence (green). Scale bar 1 µm. (D) Whole cell lysates from (C) were analysed by Western blot with anti-GFP antibodies to visualise YFP-tagged proteins. (E) The localisation of YFP-Cep63 proteins to the centrosome was scored in 3 independent experiments, n >10. (F) Overexpression of Cep63 425–541 and 136–541 deplete Cep152 from the centrosome. The ratio of Cep152 to γ-tubulin fluorescence intensities at the centrosome was measured for multiple cells from the experiment shown in (C), n >10.