PMC

CEP120 interacts with C2CD3 and Talpid3. (a) CEP120 physically associates with C2CD3 in vivo. HEK293T cells were co-transfected with CEP120-myc and GFP-C2CD3 constructs. Twenty-four hours after transfection, cell lysates were immunoprecipitated (IP) and then immunoblotted (IB) with the indicated antibodies. (b) Mapping the C2CD3-interacting domain in CEP120. HEK293T cells were co-transfected with GFP-C2CD3 and various myc-tagged CEP120 constructs, analyzed by IP, and subsequent IB using the indicated antibodies. (c) CEP120 directly interacts with C2CD3. GST and various GST-CEP120 recombinant proteins were affinity purified (left). The full-length ³⁵S-methionine-labeled C2CD3 proteins (middle) were incubated with bead-bound GST or various GST-CEP120 recombinant proteins (right) and analyzed by SDS-PAGE and autoradiography. (d) CEP120 physically associates with Talpid3 in vivo. HEK293T cells were co-transfected with CEP120-myc and GFP-Talpid3 constructs, and analyzed as described in (a). (e) Mapping the Talpid3-interacting domain in CEP120. HEK293T cells were co-transfected with GFP-Talpid3 and various myc-tagged CEP120 constructs and analyzed as described in (b). Uncropped blots of (a,b,d,e) are shown in Fig. , respectively. (f) CEP120 directly interacts with Talpid3. The full-length ³⁵S-methionine-labeled Talpid3 proteins (left) were incubated with bead-bound GST or various GST-CEP120 recombinant proteins (right) and analyzed by SDS-PAGE and autoradiography. (g) Schematic summary of the interactions of CEP120, C2CD3, and Talpid3, and the positions of the disease-associated CEP120 mutations that cause JS.

Expression and localization of Cep120. (A–C) Lysates prepared in RIPA buffer were resolved by SDS-PAGE and subjected to immunoblot analysis. (A) The Cep120 antiserum recognizes exogenous Cep120 expressed in COS7 cells and endogenous Cep120 in brain lysates. The arrow indicates the Cep120 protein. (B) Cep120 protein levels in E17.5 mouse tissues. (C) Cep120 protein levels in the mouse brain at different developmental stages. Each lane was loaded with 20 μg total proteins in (B) and (C). (D and E) Cep120 is a centrosomal protein. (D) EGFP-Cep120 colocalizes with pericentrin in cultured neocortical cells. (E) Affinity-purified Cep120 antibody labels the centrosome in cultured neocortical cells. (F and G) Expression of Cep120 in the mouse neocortex at E12.5. The Cep120 antibody prominently labels centrosomes (indicated by pericentrin co-staining) along the ventricular surface (bottom). The boxed area in (F) is shown in (G) at higher magnification to reveal the puncta labeled by both Cep120 and pericentrin antibodies along the ventricular surface. Scale bars: 10 μm in (D) and (E), 20 μm in (F) and (G).

(A) Schematic of centriole duplication and the cell cycle. Cep120 is enriched on the youngest generation of centrioles at all stages of cell division. Importantly, Cep120 is asymmetrically localized and enriched on the daughter centriole during G₀. (B) Cep120 depletion from MEF cells by siRNA transfection. Lysates were probed for Cep120 and actin (loading control). Numbers below indicate relative levels of Cep120, normalized to actin. (C) MEFs were transfected with the indicated siRNA, serum starved for 24 hr, fixed, and stained for Cep120, centrin (centrioles), Cep164 (mother centriole) and DAPI (DNA). (D) (Left) Quantification of the fraction of transfected cells with Cep120 staining at the centrosome. N = 300 (control) and 300 (Cep120) siRNA. (Right) Ratio of fluorescence intensity of centrosomal Cep120, in control versus Cep120-depleted cells. N = 214 (control) and 244 (Cep120) siRNA. (E) FACS analysis performed on MEF cells transfected with control or Cep120 siRNA, incubated in normal growth medium for 24 hr, then in low-serum medium for another 24 hr. The percentage of cells at each cell cycle phase is indicated. (F) siRNA transfected MEF were grown for 24 hr in normal growth medium (cycling), then incubated in low-serum medium (serum-starved) for 24 hr. Cells were fixed and stained with antibodies against Ki-67 to identify proliferating cells. DNA was stained with DAPI. Graphs denote the fraction of Ki-67-positive cells during cycling and serum-starvation (SS) conditions. N = 300 (control cycling), N = 300 (control SS), N = 300 (Cep120 cycling), N = 300 (Cep120 SS). All results are averages of three independent experiments; *p<0.05. Scale bars = 10 μm.

CEP120 interacts with CPAP. (A) A schematic representation of the interaction between CEP120 and CPAP. (B) Coimmunoprecipitation experiments showed that endogenous CEP120 and CPAP form a complex in vivo. (C) CPAP interacts with the C3 domain of CEP120. (D) CEP120 interacts with the CP3 domain of CPAP. (E) Mapping the CPAP-interacting domain in CEP120 by GST pull-down assays. Full-length [³⁵S]methionine-labeled CPAP proteins were incubated with bead-bound GST or various GST-CEP120–truncated proteins and analyzed by SDS-PAGE and autoradiography. (F) Mapping the CEP120-interacting domain in CPAP.