B in cellular transformation. Materials and methods Generation of Bub1 transgenic mice Bub1 transgenic mice were generated according to previously described methods. Bub1 transgenic mice were maintained on a mixed 129SV/E C57BL/6 genetic background. E-Myc mice were bred with Bub1T85 mice to generate cohorts consisting of E-Myc, and E-Myc;Bub1T85 double-transgenic mice. Mice were housed in a pathogen-free barrier environment. Mouse protocols were reviewed and approved by the Mayo Clinic institutional animal care and use committee. Mice in tumor susceptibility studies were monitored daily. Moribund mice were killed and major organs screened for overt tumors. Tumors were processed for histopathology by standard procedures. Prism software was used for the generation of tumor-free survival curves and statistical analyses. Generation and culture of MEFs Bub1 transgenic MEFs were generated and cultured as described previously. MEFs were frozen at P2 or P3 and used for MedChemExpress AZ-6102 experimentation between P4 and P6. At least three independently generated MEF lines per genotype were used unless otherwise stated. Mitotic MEFs were prepared by two methods. Mitotic MEFs were collected after culturing asynchronous cells for 4 h in medium containing 100 ng/ml nocodazole and harvesting cells by shake-off. Alternatively, cells were synchronized in G0 by serum starvation for 12 h and released into 10% FBS containing DME. 0.5 M taxol was then added 12 h after release and cells were harvested 18 h after release. Flag-tagged BubR1 was constitutively expressed using the Z/EG expression vector, similar to Bub1. Cell culture, Western blot analysis, and immunoprecipitation HeLa cells were cultured in DME supplemented PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19834545 with 10% FCS. Human cancer cell lines DOHH2, Ramos, and Raji were cultured in RPMI supplemented with 10% FCS. Western blot analysis was performed as described previously. The following antibodies were used: rabbit anti-Bub1; 12CA5 or 16B12; pSer10-H3; -actin; Aurora B; pT232-Aurora; GFP; pT121-H2A; H2A; BubR1; Bub3; and Mad2. Immunoprecipitations for Aurora B and Bub1 were performed on mitotic cell extracts prepared in RIPA buffer. Antibodies for immunoprecipitation were Aurora B and Bub1. Mitotic HeLa cells were prepared from cells that were serum starved for 12 h and released into 20% FBS containing DME. After 18 h, 0.5 M taxol was added and cells were harvested 6 h later. Indirect immunofluorescence and confocal microscopy Indirect immunofluorescence 
was performed as described previously. Cells were plated onto chamber slides and incubated for 24 h. Standard fixations for immunostainings were with 1% paraformaldehyde for 5 min at room temperature. Primary antibodies, which were incubated overnight, were as follows: rabbit anti-Bub1; human anti-centromeric antibody; rabbit antiSgo1; rabbit anti-pCenp-A; rabbit anti-pT121-H2A; rabbit anti-pKnl1; rabbit anti-Cdc20; rabbit antiMad2; mouse anti-BubR1; mouse anti-Aurora B; rabbit anti-pThr232-Aurora; and mouse anti-centrin2. A laser-scanning microscope with Axiovert 100M with a c-Apochromat 100x oil immersion objective was Oncogenic Bub1 hyperactivates Aurora B kinase Ricke et al. 1061 used to analyze immunostained cells and to capture representative images. For quantification, we used ImageJ software. Confocal images were converted to 8-bit grayscale. Cell edges were traced using the freehand tool, and the mean pixel intensity within the marked area was calculated. The integrated density, defined as the mean pi