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llected by centrifugation, washed with acetone, airdried and resolved by SDS-PAGE. Antibodies Antibodies used for Western blotting: anti-Bub1, anti-pT120-H2A and anti-a-tubulin. Antibodies used for immunofluorescence microscopy: anti-Mad1, anti-cMad2 anti-Borealin, anti-INCENP, anti-Bub1 was produced after mice were injected with Bub1 recombinant protein spanning residues 1318, anti-Bub1, CREST anti-human auto-immune serum, anti-Aurora B, anti-Bub1, anti-CENP-E, anti-Mad2, anti-Sgo1, anti-Sgo2, anti-pT120-H2A, anti-pS7CENP-A, anti-pT3-H3 and anti-pS10H3. The polyclonal MCAK antibody was raised in rabbits by immunization with bacterially expressed His-MCAKaa588-725. For immunofluorescence experiments, all primary antibodies were detected with AlexaRed-594-, AlexaRed-564-, and AlexaGreen-488labeled secondary anti-mouse and anti-rabbit antibodies or Cy5conjugated donkey antibodies. For Western blotting, signals were detected using HRP-conjugated anti-mouse or anti-rabbit antibodies. In-cell western HeLa cells were seeded into 96-well plates for 5 hr at 37C. Then, cells were treated with Nocodazole for 16 hr and varying concentrations of test compounds for 1 hr. The cells were fixed, washed and blocked with buffer before incubating with the primary antibody overnight at 28C. After washing, secondary IRDye-labeled antibody mix with cell stains was added for 1 hr and washed again. Plates were scanned with a LiCor Odyssey Infrared Imager at 800 nm for P-H2A and at 700 nm for Draq5/Sapphire, a cell stain. The signal ratio for cells treated only with Nocodazole was set to 100% and the corresponding ratio for untreated cells to 0%. The IC50 value was then determined by curve fitting. Immunofluorescence microscopy, image processing, quantification and live cell imaging For fluorescence microscopy cells were grown on coverslips and fixed in PTEMF buffer or methanol at 20C, respectively. Images of randomly selected cell were acquired as z-stacks using a DeltaVision microscope on an MedChemExpress LGX818 Olympus IX71 base, equipped with a Plan Apochromat N 60x/NA1.42 oil immersion PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19825213 objective and a CoolSNAP HQ2 camera. Serial optical sections were deconvolved and projected using SoftWorx software. Images were quantified as previously described using automated pipelines run by Cell Profiler software. Results from 23 independent experiments were pooled and statistical Baron et al. eLife 2016;5:e12187. DOI: 10.7554/eLife.12187 19 of 26 Research article Cell biology analysis was done with GraphPad Prism software. Error bars on histograms illustrate SEM. Scale bars represent 10 mm. For time-lapse imaging, cells were imaged using a Nikon ECLIPSE Ti microscope equipped with a CoolLED pE-1 illumination system and a 20x/NA0.75 air Plan Apochromat objective in a climate-controlled environment. Images were acquired at multiple positions at indicated time intervals. MetaMorph 7.7 software was used for acquisition and processing of data. FRET, FRAP, and high sensitivity microscopy experiments were carried out using a spinning disk confocal system based on a Zeiss Axio Observer stand equipped with a Photometric Evolve 512 back-illuminated EMCCD camera, 63x/NA1.4 plan apochromat objective and diode lasers and run by SlideBook software. FRET analyses were carried out by excitation with a 440 nm diode laser and by recording of CFP and YFP fluorescence emission in zstacks. Background-corrected FRET ratios were calculated in ImageJ using the Ratio Plus plugin. FRAP analysis of EGFP-Bub1 was p