Immunoprecipitation and Immunoblotting of Cell Lysates

Cells were lysed at 4°C for 30 mins in lysis buffer: 30 mM Tris HCl, 150 nM NaCl, 2 mM EDTA, 10% Glycerol, 0.2% Triton X-100, with PhosSTOP (Roche) and Complete protease inhibitors (Roche). Lysates were incubated with anti-CDC20 antibody (Abcam, 26483) for 1 hr at room temperature, then incubated a further 15 mins with dynabeads (Life technologies). The beads were washed, bound proteins eluted using 0.2 M glycine [pH 2.5] and SDS loading buffer added prior to immunoblotting on NuPAGE Tris-Acetate gels (Life Technologies) as previously described [52 (link)]. Primary antibodies used were: α-tubulin (Sigma, T9026), BUB3 (BD Biosciences, 611731), BUBR1 (BD Biosciences, 612503), CDC20 (Millipore, MAB3775), GFP (Clonetech, 632381), Histone H3 (Abcam, ab1791), Histone H3 pS10 (Millipore, 06–570), MAD2 (Bethyl Laboratories Inc., A300-301A), MPS1 (Millipore, 05–682), MPS1 pT33pS37 (Life Technologies, 44–1325G), MPS1 pT676³⁰, Cleaved PARP (Cell Signaling, 9541), Cleaved Caspase 3 (Cell Signaling, 9661), p53 (Thermo Fisher Scientific, MS-738-P).