Figure 4.

CYLD-mediated deubiquitination of Cep70 promotes its interaction with γ-tubulin and localization at the centrosome. (A) Immunoprecipitation (IP) and immunoblotting (IB) showing the interaction between endogenous CYLD and Cep70 in RPE-1 cells, by using either the total cell lysate or the centrosomal preparation. (B) Examination of Cep70 ubiquitination by GST pull-down (PD) and immunoblotting in cells transfected with control or CYLD siRNAs, together with His-Myc-ubiquitin and GST-Cep70. (C) Immunoblots showing that transfection of GFP-CYLD, but not its C/S mutant, rescues the level of Cep70 ubiquitination in CYLD-depleted cells. (D) Examination of Cep70 ubiquitination in cells transfected with control or CYLD siRNAs, together with His-Myc-ubiquitin and GFP-Cep70 or its mutants. ΔCC1, without the first coiled-coil domain. ΔCC2, without the second coiled-coil domain. 15KR, mutation of all the 15 lysines in the CC2 domain to arginines. (E) Immunofluorescence images of cells transfected with GFP-Cep70 or its mutants, followed by staining with anti-γ-tubulin antibody and DAPI. Scale bar, 5 μm. (F) Quantification of the relative centrosomal localization of Cep70 or its mutants in cells treated as in E. n= 30. Experiments were performed 3 times. (G) Immunoprecipitation and immunoblotting showing the interaction of γ-tubulin with GFP-Cep70 and its 15KR mutant in RPE-1 cells transfected with control or CYLD siRNAs. (H) Immunofluorescence images of RPE-1 cells transfected with CYLD siRNA and GFP, GFP-Cep70, or GFP-Cep70-15KR and serum-starved for 48 h, followed by staining with anti-ace-tubulin antibody and DAPI. Scale bar, 5 μm. (I-K) Relative centrosomal localization of GFP-Cep70 and its 15KR mutant (I, n = 30), percentage of ciliated cells (J, n = 50), and ciliary length (K, n = 30) in cells treated as in H. Experiments were performed 3 times. Student's t test for all graphs. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001; ns, not significant. Error bars indicate SEM.