Nedd1

The following antibodies were used for immunoblotting or immunoflourescence; PLK1 (Invitrogen 33-1700; 1:1000), CREST (Europa, 1:1000), Pericentrin (Abcam 4448; 1:500) β-tubulin (Sigma-Aldrich D66; 1:1000), GFP (Clontech JL-8 632381; 1:1000), V5 (Invitrogen R96025; 2ul for IP & 1:5000 for immunoblotting) PhosphoS10-histone H3 (Abcam 14955; 1:2500), Nedd1 (Abcam 57336; 1:1000), β-actin (Sigma-Aldrich A5441; 1:10000), HRP-conjugated Mouse/Rabbit/ L-chain specific (Jackson ImmunoResearch Laboratories, Inc., 1:10000); Alexa Fluor^TM mouse 568, 594, human 594 and rabbit 633 secondary antibodies used for immunofluorescence (ThermoFisher Scientific; 1:500).

Rabbit antibodies against tubulin C102 were a gift of Dr. M. Andreu and HEC1 antibodies were a gift of Dr. PT. Stuckenberg (University of Virginia, Charlottesville, VA, USA). Polyclonal antibodies against TPX2 and PolyE-Tubulin were home-made [32 (link),33 (link)]. All other antibodies are commercially available: Acetyl-tubulin 6-11B-1 (Santa Cruz, Santa Cruz, CA, USA), α-tubulin DM1A (Sigma, Burlington, MA, USA), Vinculin (Sigma), γ-tubulin GTU88 (Sigma), Kinesin 5 (Abcam, Cambridge, UK), pericentrin (Abcam), NEDD1 (Abcam), PRC1 (Santa Cruz), HURP (Proteintech, Chicago, IL, USA), CREST (Europa Bioproducts, Cambridge UK), PLK1 clone 35-206 (Millipore, Burlington, MA, USA), KIF2A (Novus, Littleton, CO, USA), NuMA (Abcam), BUBR1 (Cell Signaling, Danvers, MA, USA), MCAK (Millipore), MYPT1 (Proteintech).
Most chemicals, including Nocodazole, STLC, and MG132, were obtained from Sigma. BI2356 was purchased from MedChemExpress.

HCT116-PLK1 cells were treated with TET induction and/or cantharidin and fixed with 100% cold methanol for 10 minutes and permeabilized in 0.25% Triton X-100. Antibody incubation was carried out in 1% BSA in PBS for 1 hour at 37°C with the following antibodies from Abcam: NEDD1 (abcam57336), β-tubulin (abcam6046), CENPA (abcam13939). The γH2AX antibody (2607372) was obtained from EMD Millipore. Primary antibodies were detected with Alexafluor-594 and Alexafluor-488-conjugated secondary antibodies (Life Technologies, A11005, A11034). DNA was stained using DAPI. Three dimensional image stacks of mitotic cells were acquired in 0.2μm steps using a 63X oil-immersion objective on an FV300 confocal laser scanning biological microscope (Olympus). Image stacks were deconvolved using Fiji software. Centromere distance measurements were made manually using FV10-ASW 4.0 Viewer.