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Fig. 3 | BMC Biology

Fig. 3

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

Fig. 3

Astrin protects HCR from ubiquitination and ensures the centrosome localization of HCR. A Negative control, astrin, and HCR siRNA-treated HeLa cells were analyzed by western blotting with antibodies against HCR, astrin, and beta-actin. The relative abundance of HCR protein was normalized to beta-actin and statistically analyzed. Error bars represent the mean ± SD of three independently performed experiments (n = 3); **P < 0.01 and ***P < 0.001 (Student’s t test). The individual data values were provided in Additional file 6: Raw Data. B HeLa cells transfected with GFP alone or GFP-astrin were immunoblotted for GFP, HCR, and beta-actin. The relative protein levels of HCR were statistically analyzed across three independent experiments (n = 3). Error bars represent the mean ± SD; ***P < 0.001 (Student’s t test). The individual data values were provided in Additional file 6: Raw Data. C GFP alone or GFP-astrin-transfected HeLa cells were subjected to immunostaining with HCR (red), gamma-tubulin (cyan), and DAPI (blue); scale bars, 10 μm; inset scale bars, 1 μm. The relative intensity of HCR in the centrosome was normalized to gamma-tubulin and statistically analyzed. Error bars represent the mean ± SD; **P < 0.01 (Student’s t test). D Negative control, astrin, or HCR siRNA-treated HeLa cells were co-stained with HCR (red), gamma-tubulin (green), and DAPI (blue); scale bars, 10 μm; inset scale bars, 1 μm. The relative intensity of HCR in the centrosome was normalized to gamma-tubulin and statistically analyzed. One hundred cells (n = 100) per group were counted for each condition from three independent experiments. Error bars represent the mean ± SD; ***P < 0.001 (Student’s t test). E Negative control, astrin, and HCR siRNA-treated HeLa cells were co-stained with astrin (red), gamma-tubulin (green), and DAPI (blue); scale bars, 10 μm; inset scale bars, 1 μm. The relative intensity of HCR in the centrosome was normalized by gamma-tubulin and statistically analyzed. One hundred cells (n = 100) per group were counted for each condition from three independent experiments. Error bars represent the mean ± SD; ***P < 0.001; ns, no significance (Student’s t test). F Real-time PCR analysis of HCR mRNA levels in control and astrin siRNA-transfected HeLa cells. For statistical analysis, HCR mRNA levels were normalized to GAPDH; n = 4; ns, no significance (Student’s t test). The individual data values were provided in Additional file 6: Raw Data. G Parental and astrin-KO HeLa cells were treated with MG132 or DMSO for 16 h to suppress the ubiquitin-proteasome pathway. The lysates of each group were analyzed by immunoblotting with antibodies to HCR, astrin, ubiquitin, GAPDH, and beta-actin. For quantitative analysis, the level of HCR of each group was normalized to beta-actin. Error bars represent the mean ± SD of three independently performed experiments (n = 3); **P < 0.01; ns, no significance (Student’s t test). The individual data values were provided in Additional file 6: Raw Data. H mCherry empty vector or HCR-mCherry plasmid in conjunction with ubiquitin-HA was transfected into parental or astrin-KO HeLa cells for immunoprecipitation. The precipitates were analyzed by western blot with the indicated antibodies. I DMSO- or MG132-treated parental HeLa cells and astrin-KO HeLa cells were co-stained with HCR (red), gamma-tubulin (green), and DAPI (blue); scale bars, 10 μm

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