Fig. 5: Endogenous SMC2 T574 is phosphorylated by AURKA in mitosis in pre-rRNAs-depleted cells.

A Dot blotting was performed with non-T574 phospho-SMC2 peptide or T574 phospho-SMC2 peptide to detect the specificity of anti-SMC2 T574-P antibody. B HeLa cells were treated with the indicated reagents. These cells were synchronized at M phase by thymidine double blocking and harvested by shaking off. Proteins were resolved by SDS-PAGE and probed with the indicated antibodies. Alpha-tubulin was used as a loading control. C HeLa cells were transfected with the indicated plasmids and treated with BMH-21 or not. These cells were synchronized at M phase by nocodazole and harvested by shaking off. Co-immunoprecipitation was performed with anti-Flag antibody, and the phosphorylation levels of Flag-SMC2 were evaluated by western blot using anti-SMC2 T574-P antibody. Alpha-tubulin was used as a loading control. D HeLa cells were transfected with the indicated siRNAs and treated with BMH-21 or not. These cells were synchronized and harvested as described in (C). Proteins were resolved by SDS-PAGE and probed with the indicated antibodies. Alpha-tubulin was used as a loading control. E HeLa cells were transfected with the indicated doses of Flag-AURKA or Flag vector and treated with BMH-21 or not. These cells were synchronized and harvested as described in (C). Proteins were resolved by SDS-PAGE and probed with the indicated antibodies. Alpha-tubulin was used as a loading control. F HeLa cells were treated with the indicated doses of TCS7010 or MLN8237, and treated with BMH-21 or not. These cells were synchronized and harvested as described in (C). Proteins were resolved by SDS-PAGE and probed with the indicated antibodies. Alpha-tubulin was used as a loading control. G HeLa cells were treated with Act D, BMH-21, or DMSO. These cells were fixed, and indirect immunofluorescent staining was performed using anti-SMC2 T574-P antibody. Chromosomes were stained by DAPI. Scale bar, 8 μm.