Cdc27

Immunoblotting was performed as previously described [42 (link)], except that a ChemiDoc Touch imaging system (Bio-Rad, Hercules, CA, USA) was used to detect the signals. The following antibodies were obtained from the indicated sources and used at the indicated concentrations: monoclonal antibodies against beta-actin (Sigma-Aldrich; 0.2 µg/ml), FLAG (Sigma-Aldrich; 1 µg/ml), BUB3 (BD Biosciences, Franklin Lakes, NJ, USA; 0.25 µg/ml), CDC27 (BD Biosciences; 0.125 µg/ml), cleaved PARP1(Asp214) (BD Biosciences; 0.2 µg/ml), CDH1 (Thermo Scientific; 1 µg/ml), CDC20 (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 0.2 µg/ml), MAD1 (Santa Cruz Biotechnology; 0.2 µg/ml),TRIP13 (Santa Cruz Biotechnology; 0.2 µg/ml), APC4 (Abcam, Cambridge, UK; 0.1 µg/ml), polyclonal antibodies against phosphor-histone H3^Ser10 (Santa Cruz Biotechnology; 0.1 µg/ml), BUBR1 (Bethyl Laboratories, Montgomery, TX, USA; 0.2 µg/ml), MAD2 [43 (link)], and p31^comet [44 (link)]. Antibodies against cyclin B1 (1 µg/ml) were gifts from Julian Gannon (Cancer Research UK). Immunoprecipitation of MAD2 or CDC20 was performed as previously described [43 (link)]. Antibody against APC4 for immunoprecipitation was prepared by immunizing rabbits with an APC4 peptide (CIVIKVEKLDPELDS) (GenScript, Piscataway, NJ, USA).

Total cell lysates were prepared by lysing 10∧6 cells in 100 µl SDS-sample buffer containing 5% 2β-mercaptoethanol. After boiling, 10 µl were separated by denaturing gel electrophoresis transferred to nitrocellulose membrane using semi-dry electroblotting and analysed by using antibodies against CDC27 (BD Transduction Lab, Vienna, Austria), α-tubulin (Tat-1, J. Gannon, Cancer Research UK, South Mimms, UK), BIM (BD Bioscience, Vienna, Austria), or GAPDH (mAb6C5, HyTest. Ltd., Turku, Finland) followed by enhanced chemiluminescence using HRP-conjugated secondary antibodies.