Fig. 5

CCP5 interacts with CP110 through its N-terminus. A The lysates of HEK293T cells stably expressing myc-tagged LacZ or CCP5 were immunoprecipitated with anti-myc affinity beads. Protein levels of endogenous CP110 and myc-CCP5 in immunoprecipitants and cell lysates were detected. CP110 can be immunoprecipitated from cells expressing myc-CCP5 but not myc-LacZ. B Lysates of control or HEK293T cells stalely expressing CCP5 were immunoprecipitated with CP110 or CEP97 polyclonal antibodies. CCP5 can be co-immunoprecipitated with CP110, but not CEP97. As a control, CP110 was co-immunoprecipitated with CEP97. C Direct interaction between CCP5 and endogenous CP110 detected by GST pull-down assay. HEK293T cell lysates were incubated with glutathione resin bound with His-GST or His-GST-CCP5 proteins. The eluent obtained by GSH competition was examined using CP110 antibody. D Schematic representation of full-length CCP5 and the indicated CCP5 truncations. ND, N-domain; CP: carboxypeptidase domain. E The myc-LacZ or full-length or truncated CCP5 was expressed in HEK293T cells and immunoprecipitated with anti-myc affinity beads. The interaction with endogenous CP110 was determined by indicated antibodies. An unspecific band from IP appears at the position marked with *. F hTERT-RPE1 cells transfected with GFP, CCP5-GFP, or CCP5 truncations with GFP tag were serum-starved for 24 h and immunostained with GFP (green) and ARL13B (red) and nuclei were visualized with DAPI. Representative images showed that similar to CCP5 wild-type, its N-terminal, but not C-terminal truncation is sufficient to inhibit cilia formation. G Quantification of the ciliation in GFP-positive cells shown in F (3 independent experiments; at least 30 cells analyzed per experimental condition, Additional file 2)