ted 
if purchase TMS UBASH3B regulates the error correction function of Aurora B. Sixty minutes after release from monastrol-induced monopolar prometaphase, we observed a higher number of cells in prometaphase, a lower number of cells in metaphase and telophase and no difference in anaphase upon downregulation of UBASH3B as compared to the control siRNA-treated cells. Specifically, downregulation of UBASH3B led to a lower number of monopolar prometaphases, a higher number of bipolar prometaphases and a lower number of metaphases, confirming that UBASH3B does not regulate bipolar spindle formation but may control the correction of KT-MT attachments during mitotic progression. Moreover, cold treatment of metaphase cells showed a decrease in the formation of stable kinetochore bundles upon UBASH3B depletion and a profound reduction of the kinetochore levels of Astrin. Collectively, these results suggest that UBASH3B also regulates the function of Aurora B at the centromere. The defects in kinetochore attachment may prevent SAC satisfaction which explains the delay in anaphase onset and death in prometaphase observed upon downregulation of UBASH3B. Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscripts Dev Cell. Author manuscript; available in PMC 2017 April 21. Krupina et al. Page 9 UBASH3B forms a functional complex with MKlp2 to target Aurora B to microtubules prior to anaphase Next, we addressed the precise molecular mechanisms underlying UBASH3B-mediated microtubule targeting of Aurora B. Since the mitotic kinesin-like protein 2 kinesin was demonstrated to mediate the midzone localization of Aurora B, we hypothesized that it may act in a complex with UBASH3B prior to anaphase to shift the balance of Aurora B towards microtubules. Moreover, MKlp2 was previously found on the spindle microtubules in pre-anaphase cells and UBASH3B localized preferentially to the prometa- and metaphase spindles. To test whether UBASH3B interacts with MKlp2, we immunoprecipitated GFP-tagged UBASH3B or GFP alone from the extracts of cells arrested in mitosis by Taxol. Endogenous MKlp2 visibly interacted with GFP-UBASH3B but not with GFP control, suggesting that a functional complex of Aurora B, MKlp2 and UBASH3B may exist in pre-anaphase cells. Consistent with the previous findings, we found a slower migrating, modified form of Aurora B to interact with UBASH3B. In contrast, UBASH3B bound to a presumably unmodified form of MKlp2 protein, suggesting that the UBASH3B-MKlp2 interaction is not dependent on ubiquitin recognition. To further corroborate this finding, we overexpressed GFP-tagged UBASH3B in cells arrested in prometaphase by PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19811403 STLC in the presence of MG132 and analyzed localization of MKlp2. Overexpression of UBASH3B was sufficient to target MKlp2 to monopolar spindles. The spindles contained almost twice of the amount of MKlp2 protein as compared to the GFP-control expressing cells. Moreover, inhibition of MKlp2 motor activity using paprotrain led to dissociation of MKlp2 from the mitotic spindle and accumulation of UBASH3B around the centrosomes, in contrast to the control cells, which displayed uniform distribution of UBASH3B along the entire mitotic spindles. In many cells, paprotrain treatment abolished microtubule localization of UBASH3B, suggesting that MKlp2 and UBASH3B are mutually co-dependent for their spindle function. Accordingly, inactivation of UBASH3B by siRNA prevented microtubule and midbody association of MKlp2 in metaphase and