in the presence or absence of microtubules. We conclude that microtubules can stimulate active kinase in vitro. EB1 and Aurora B can coimmunoprecipitate in HeLa cells. To confirm that Aurora B can directly interact with EB1, we purified full-length Xenopus EB1 and Xenopus Aurora B bound to a C-terminal fragment of Xenopus INCENP that includes the IN-box from E. coli. AI790856 bound to beads could pull down EB1 in a concentrationdependent manner. To identify the subcellular location of the interaction between EB1 and Aurora B, we performed a proximity ligation in situ assay using anti-EB1 and antiAurora B antibodies. PLA is an antibody-based technique that allows the visualization of two proteins only if they are in close proximity with high sensitivity. Standard PLA was performed to identify the subcellular locations where Aurora B was in close proximity to tubulin. We used Xenopus S3 cells for two reasons. First, these cells possess a normal karyotype and remain very flat in mitosis to provide outstanding imaging. Second, our Xenopus Aurora B antibodies are highly specific and provide very reproducible signals in the PLA assay. We performed PLA for Aurora Btubulin, and then, the cells were further processed by standard immunofluorescence with antibodies directly conjugated with fluorophores to tubulin and INCENP to generate fiducial marks on the spindle. The PLA signal in prometaphase cells could be detected at inner centromeres and adjacent to inner centromeres on microtubules. This is similar to the EB1Aurora B interactions seen by PLA and is consistent with our observation that EB1/microtubules localize Aurora B to inner centromeres. In addition, we detected an additional PLA signal throughout the spindle. Cells were subjected to a brief ice treatment to destabilize nonkinetochore-associated microtubules. We observed a significant drop in PLA signal in ice-treated prometaphase cells, including most kinetochores that were not situated near centrosomes. Some kinetochore/centromere PLA signals persisted in metaphase, suggesting that Aurora B can interact with K-fibers. However, most of the metaphase signals throughout the DMXB-A spindle were 
cold sensitive, suggesting that Aurora B can interact with astral microtubules. To verify the specificity of the PLA signals, we performed parallel assays in which the antiAurora B antibody was omitted and the PLA signal was greatly reduced. We hypothesized that the interactions between EB1 Aurora B and tubulinAurora B that we visualize at prometaphase kinetochores were with preK-fibers. This hypothesis is strongly supported by the observation that inner centromeric Aurora B levels are similar in cells treated with 0.33 M nocodazole- and DMSO-treated prometaphase cells. In addition, after cold PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19833994 treatment, the bulk of Aurora B tubulin interactions next to 953 EB1 and microtubules control Aurora B recruitment Banerjee et al. 954 JCB VOLUME 204 NUMBER 6 2014 prometaphase kinetochores were lost. PreK-fibers are small bundles of microtubules that protrude from kinetochores before they make mature attachments and can mediate lateral attachments between dynein on kinetochores and microtubules. PreK-fibers are difficult to distinguish in a whole spindle, but they are readily visualized in monastrol and after cells are washed out of nocodazole. Xenopus S3 cells were incubated in monastrol, and cells were stained for xNdc80 to visualize kinetochores, tubulin to visualize the spindle, and PLA to detect where Aurora
