Cdc20

The following antibodies were used for Western blotting: APC11 (1/500, ab57158) and MYC (1/5,000, ab32072), Abcam; APC3 (1/1,000, 610454), cyclin A (1/1,000, 611269), Mcl‐1 (1/500, 559027) and Nek2A (1/500, 610593), BD Transduction Laboratories; APC8 (1/1,000, A301‐181A) and Fbw7 (A301‐720A‐1), Bethyl Laboratories; APC2 (1/1,000, 12301S) and cyclin E (1/1,000, 4129S), Cell Signaling Technologies; MPM2 (1/1,000, 05‐368), Millipore; APC10 (1/1,000, sc‐166790), Cdc20 (1/1,000, sc‐13162) and cyclin B1 (1/2,000, sc‐752), Santa Cruz Biotechnology; Actin (1/4,000, A2066) and Mad2 (1/1,000, PA5‐21594), Sigma Aldrich; Cdh1 (1/500, MS1116‐P0), Thermofisher. Antibodies were used according to standard protocols except for Fbw7 where blocking and primary antibody incubation were carried out in ReliaBLOT (Bethyl Laboratories). Histone H3 phospho‐Ser10 (05‐806), Millipore, was used for flow cytometry as described previously (Harley et al, 2010). Fbw7 (A301‐721A‐1), Bethyl Laboratories, was used for immunoprecipitation. Doxycycline, thymidine, paclitaxel and reversine were purchased from Sigma Aldrich. Nocodazole (Calbiochem) was routinely used at 830 nM apart from assaying cyclin B1 degradation where it was used at 33 nM. ProTAME was initially provided by Dr Randall King (Dept. of Cell Biology, Harvard University) and subsequently purchased from Boston Biochem.

Tissue/cell protein lysates were generated by SDS (1%, m/v) lysis buffer. These lysates were equally loaded on a 5% and 10% gel, electrophoresed and transferred to enhanced cheiluminescence-nitrocellulose membranes (Amersham). For detection of protein, the membranes were incubated with antibodies against β-actin (1:10000, Merck-Millipore), Anapc2 (1:1000, Santa Cruz), CyclinB1 (1:1000, Santa Cruz), Cdc20 (1:1000, Santa Cruz), p27 (1:500, Cell Signaling Technology), Skp2(1:1000, Abcam), Cdk2(1:1000, Abways), cyclin E(1:500, Abways), p21(1:300, Santa Cruz), at 4°C overnight, followed by incubation with HRP-conjugated secondary antibody (1:20000, Cell Signaling Technology) at RM for 1 h. The detection of protein signal was performed by a SuperSignal West Pico Chemiluminescent Substrate Kit (Pierce, Rockford).

Whole protein extracts were obtained by lysing cells in TNN buffer (50mM Tris (pH 7.5), 120mM NaCl, 5mM EDTA, 0.5% NP40, 10mM Na4P₂O₇, 2mM Na₃VO₄, 100mM NaF, 1mM PMSF, 1mM DTT, 10mM β-glycerophosphate, protease inhibitor cocktail (Sigma)). Protein lysates or purified protein were separated by SDS-PAGE, transferred to a PVDF-membrane and detected by immunoblotting with the first and secondary antibodies: β-actin (Santa Cruz Biotechnology, sc-47778) 1:5000, B-MYB (clone LX015.1, [42 (link)] 1:5, anti-HA.11 (Covance, MMA-101P) 1:1000, anti-FLAG M2 (Sigma, F3165) 1:5000, anti-His (Sigma, H1029) 1:2000, Vinculin (Sigma, V9131) 1:10000, TOP2A (Santa Cruz Biotechnology, sc-365916) 1:1000, CDC20 (Santa Cruz Biotechnology, sc-5296) 1:500, YAP (Santa Cruz Biotechnology, sc-10199) 1:1000, p-YAP(S127A) (Cell Signaling Technology, 4911) 1:1000, LIN9 (Bethyl, A300-BL2981), NUSAP1 (Geert Carmeliet) 1:1000, CENPF (Abcam, ab-5) 1:1000, anti-mouse-HRP (GE healthcare, NXA931) 1:5000 and HRP Protein A (BD Biosciences, 610438) 1:5000. For immunoprecipitation of FLAG-tagged proteins, protein G dynabeads (Thermo Fisher Scientific) were first coupled with 1 μg FLAG-antibody (Sigma, F3165) and then immunoprecipitated with 1mg whole cell lysate. After five times of washing with TNN, proteins were separated by SDS-PAGE and detected by immunoblotting using the desired antibodies.

40 μg of cell lysates obtained from ground-frozen tissue or cells were used for assessing protein expression. Primary antibodies used for detection were FOXM1 (1:1000, Abcam, ab207298), MAD2 (1:1000, BD biosciences, 610678), HA (1:1000, Covance, MMS-101R), Cyclin B (1:200, Santa Cruz, SC-245), Cleaved-Caspase 3 (1:1000, Cell signaling, 9661), p-Histone H2AX (1:250, Santa Cruz, SC-517348), CDC20 (1:250, Santa Cruz, SC-13162), Actin (1:3000, Sigma, A2066). Protein band quantification was carried out using ImageJ.

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).