Mps1 pt33ps37

For immunofluorescence, cells were pretreated with CCT271850 for 1 h, then treated with nocodazole, MG132 and CCT271850 for additional 1 h. Cells were fixed and stained according to the protocol previously described (Gurden et al, 2015 (link)). Primary antibodies used were as follows: anticentromere antibodies (ACA; ImmunoVision, Springdale, AR, USA, HCT-0100), BubR1 (BD Biosciences, San Jose, CA, USA, 612503), Mad1 (Abcam, Cambridge, UK, ab45286), Mad2 (Bethyl Laboratories Inc., Montgomery, TX, USA, A300-301A), MPS1 (Invitrogen, 35–9100), MPS1 pT33pS37 (Life Technologies, 44–1325 G) and Zwint-1 (Abcam, ab84367). Images were acquired using a Zeiss LSM 710 confocal microscope and processed using the Volocity 3D Image analysis software (PerkinElmer). Time-lapse microscopy was performed in 96-well Ibidi plate (Thistle Scientific, Glasgow, UK) using a Diaphot inverted microscope (Nikon, Tokyo, Japan), in a humidified CO₂ chamber at 37 °C, using a motorised stage (Prior Scientific, Cambridge, UK), controlled by Simple PCI software (Compix, Irvine, CA, USA).

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).

Analysis by immunofluorescence and time-lapse microscopy were performed as previously described [23 (link)]. When using NDGA in immunofluorescence experiments, the cells were fixed for 20 mins in ice-cold methanol. Primary antibodies used were: α-tubulin (Sigma, T9026), ACA (ImmunoVision, HST-0100), BUB1 (Abcam, ab54893), BUBR1 (BD Biosciences, 612503), CENP-A pS7 (New England Biolabs, 2187S), CENP-E (Abcam, ab5093), MAD1 (Abcam, ab45286), MAD2 (Bethyl Laboratories Inc., A300-301A), CDC2020 (Millipore, MAB3775), hDIC (Abcam, ab23905), MPS1 (Millipore, 05–682), MPS1 pT33pS37 (Life Technologies, 44–1325G), GFP (Abcam, ab6556), HEC1 (Abcam, ab3613), Histone H3 pS10 (Millipore, 06–570), KNL1 (Bethyl Laboratories Inc., A300-805A), SPINDLY (Abnova, H00054908), ZW10 (Abcam, ab21580), ZWINT1 (Abcam, ab84367).