Skip to main content
Fig. 4 | BMC Biology

Fig. 4

From: CCHCR1-astrin interaction promotes centriole duplication through recruitment of CEP72

Fig. 4

HCR directly binds to and ensures the centrosomal localization of CEP72. A Co-immunoprecipitation analysis of HCR binding to CEP72. HeLa cell lysates were immunoprecipitated with CEP72, HCR, or control rabbit IgG antibodies and analyzed by western blotting with anti-CEP72 and anti-HCR antibodies. Beta-actin was used as a negative control (left penal). GFP alone or GFP-HCR-CC3 plasmid was transfected into HeLa cells and immunoprecipitated using an GFP antibody. The precipitates were detected by immunoblotting with antibodies to GFP and CEP72 (right panel). B In vitro binding assay of HCR coiled-coil domains with CEP72. GST alone, GST-tagged CEP72, and His-tagged HCR fragments were purified from E. coli strain BL21(DE3), and a pull-down assay was performed to examine the CEP72-binding domain in HCR. C HeLa cells released from double-thymidine arrest were harvested at each time point and were analyzed by immunoblotting with antibodies against HCR, astrin, CEP72, cyclin B1, cyclin E, HURP, and beta-actin. D Negative control, CEP72 siRNA-treated HeLa cells, astrin-KO cells, and HCR-KO cells were co-stained with CEP72 (red), gamma-tubulin (green), and DAPI (blue); scale bars,10 μm; inset scale bars, 1 μm. For quantitative analysis, the intensity of CEP72 at the centrosome was normalized by gamma-tubulin. One hundred cells (n = 100) per group were counted from three independent experiments. Error bars represent the mean ± SD. ***P < 0.001 (Student’s t test). E Negative control or CEP72 siRNA-treated HeLa cells were co-stained with HCR (red) and gamma-tubulin (green) antibodies and DAPI (blue) for nuclear staining (upper panel) or co-stained with astrin (red) and gamma-tubulin (green) antibodies and DAPI (blue) for nuclear staining (lower panel). For quantitative analysis, the intensity of HCR (upper panel) or astrin (lower panel) at the centrosome was normalized to gamma-tubulin. One hundred cells (n = 100) per group were counted from three independent experiments. Each bar represents the mean ± SD (upper panel); ns, no significance (Student’s t test); scale bars, 10 μm; inset scale bars, 1 μm

Back to article page