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. Author manuscript; available in PMC: 2019 Oct 1.
Published in final edited form as: Free Radic Biol Med. 2018 Aug 13;126:358–371. doi: 10.1016/j.freeradbiomed.2018.08.009

Figure 5:

Figure 5:

NQO1-SIRT2 axis regulates APC/C complex during mitosis. (A) Cell extracts from control shRNA (sh ctr) and NQO1 shRNA (sh NQO1) MCF-7 cells were used to immunoprecipitate endogenous Cdc27. Interaction with several components of the APC/C complex was determined by western blotting using the indicated antibodies. Specificity was confirmed by using species-matched control IgG as a negative control. Input levels of the interacting proteins are shown (right). (B) MCF-7 cells overexpressing HA-Cdh1 were transfected with either control (ctr) siRNA or NQO1 siRNA followed by immunoprecipitation using an anti-acetylated lysine antibody (Ac-K). Acetylated levels of Cdh1 were checked by western blotting using an anti-HA antibody. Specificity was confirmed by using species-matched control IgG as a negative control. In parallel, immunoprecipitation with an antibody against Cdc27 was performed to check interaction between Cdc27 and Cdh1. Input levels of both Cdc27 and NQO1 are shown. (C) MCF-7 cells were transfected with either control (ctr) siRNA or NQO1 siRNA followed by immunoprecipitation using an anti-acetylated lysine antibody (Ac-K). Acetylated levels of endogenous Cdh1 were checked by western blotting using an anti-Cdh1 antibody. Specificity was confirmed by using species-matched control IgG as a negative control. In parallel, immunoprecipitation with an antibody against Cdc27 was performed to check interaction between Cdc27 and Cdh1. Input levels of Cdh1, Cdc27 and NQO1 are shown. (D) Western blot analysis in whole cell lysates MCF-7 cells transfected with either control (ctr) siRNA or NQO1 siRNA. Antibodies against well–established mitotic proteins including Aurora A, Cyclin B1 and Cdc20 were used. Mitotic cells following nocodazole treatment were collected by shake-off, replated and harvested at indicated time points. Protein levels of SIRT2, NQO1 and actin are shown. (E) Quantification of Aurora A protein levels in (D) using the ImageJ software is presented. (F) For in vivo ubiquitination assay, Myc-Aurora A and HA-Ubiquitin were co-transfected into control shRNA (sh ctr) and NQO1 shRNA (sh NQO1) MCF-7 cells. To block proteasomal degradation, cells were treated with MG132 (10 µM, 4 h). Aurora A was immunoprecipitated using an anti-Myc antibody followed by western blotting against HA to detect ubiquitinated levels of Aurora A. Levels of NQO1 and actin are shown. (G) A similar in vivo ubiquitination assay was performed in MCF-7 cells transfected with either a control (ctr) vector or a Flag-NQO1 vector. Aurora A was immunoprecipitated using an anti-Myc antibody followed by western blotting against HA to detect ubiquitinated levels of Aurora A. Levels of Flag-NQO1 and actin are shown.