Phospho-CENPA (Ser7) Antibody - #AF2330

Figure 3. CYLD regulates Aurora B activity and function. (A) HeLa cells transfected with CYLD or control plasmid were synchronized by nocodazole for 18 h before harvested. Mitotic HeLa cells were collected by mitotic shake-off. The collected cells were then continued to incubate for the indicated time points (0, 1, 3 h) and following immunoblotted with indicated antibodies. (B) HeLa cells transfected with control or Flag-CYLD plasmid were synchronized by nocodazole for 18 h and then were fixed for IF analysis with indicated antibodies. Scale bar represents 2 μm. (C) Luciferase control and 2 clones of CYLD knock-down stable HeLa cells were generated and harvested for WB assay with indicated antibodies. (D) Luciferase control or CYLD knock-down stable HeLa cells generated above were synchronized by nocodazole for 18 h and then fixed for IF analysis with indicated antibodies. Scale bar represents 2 μm. (E) HeLa cells expressing H2B-GFP were transfected with control or CYLD plasmid; 36 h later, cells were imaged at every 5 min. Around 100 cells in each group were randomly chosen for statistics. The upper panel showed the representative fluorescence video microscopy series from the onset of mitosis to monitor chromosome dynamics and the lower panel displayed the statistical results. Cell numbers were 123 (Control) and 123 (CYLD). P value was calculated using unpaired T-test. Error bars represent the standard deviation. Scale bar represents 5 μm. (F) Control and CYLD stable knock-down SiHa cervical cancer cells were plated and cultured for 24 h; cells were then stained with Hoechst. Later, more than 30 random pictures of every group were captured under a confocal microscope. Finally, we counted and summed up the proportion of cells with micronuclei. The left panel showed a representative micronuclei microscopy of the cells and the right panel displayed the statistical results. For each group, the cell number is more than 1000. P value was calculated using unpaired T-test. Scale bar represents 2 μm.