Fig. 4.

Cep57 is an anchor of the PACT domain of PCNT. a Schematic of Cep57 and the deletion mutants used for co-IP assays. Coiled-coil domains are shown in gray box; the PINC motif in pink line. The region of Cep57 for PCNT-binding is represented in red. The right column shows a summary of the co-IP results. b HEK293T cells co-expressing FLAG empty (control) or FLAG-PCNT and HA-Cep57 were immunoprecipitated (IPed) with FLAG antibodies. c HEK293T cells co-expressing FLAG-PCNT and HA-Cep57 or the indicated deletion mutants were IPed with FLAG antibodies. d Schematic of PCNT and the deletion mutants used for co-IP assays. Coiled-coil domains are shown in gray box, and the PACT domain in blue box. The region of PCNT for Cep57-binding is represented in red. The right column shows a summary of the co-IP results. We noticed that the N-terminal half of PCNT (a.a. 1–1962), which does not contain the PACT domain, also interacted with Cep57. e HEK293T cells co-expressing HA-Cep57 and FLAG-PCNT or the indicated deletion mutants were IPed with FLAG antibodies. f MBP pull-down assay showing the interaction between Cep57 and PCNT fragments in vitro. These bacterially purified recombinant proteins contain the interaction regions that were identified by the co-IP experiments in c and e. Inputs (lane 1 for Coomassie blue staining, 13.3%; lane 1 for western blotting, 1/30,000 volume of lane 1 in the Coomassie blue staining) and affinity-purified protein complexes (lanes 2–5) were subjected to SDS-PAGE, stained (Coomassie blue staining), and analyzed by western blotting. g HeLa cells co-expressing HA empty (control) or HA-Cep57 and GFP or GFP-PCNT 3113–3336 were immunostained with antibodies against HA (red), GFP (green). h HeLa cells were treated with control siRNA or siCep57, followed by transfection with the GFP-PCNT 3132–3226. The cells were immunostained with antibodies against GFP (green), Cep57 (red), and Cep192 (cyan). i Histograms represent quantification of the signal intensity of GFP and Cep57 at old mother centrioles in h (siControl n = 41, siCep57 n = 39). All scale bars, 5 μm. **p < 0.01