Mouse anti plk1

Western blotting was carried out as described23 (link). Phos-tag SDS–polyacrylamide gel electrophoresis gels were made from 7% acrylamide (37.5:1 crosslinking ratio), 70 μM Phos-tag reagent (Wako Pure Chemical Industries) and 140 μM MnCl₂, and were processed according to the manufacturer’s manual. Proteins were detected using the following primary antibodies: mouse anti-GFP (1:1,000, Roche 11814460001), mouse anti-PLK1 (1:2,000, Santa Cruz sc-17783), mouse anti-histone H3 pSer10 (1:5000, Abcam 14955), mouse anti-α-tubulin (1:5000, Sigma-Aldrich T9026), rabbit anti-phospho-(Ser) CDK substrate (1:1000, Cell Signaling 2324), rabbit anti-histone H3 (1:2000, Abcam ab1791), mouse anti-FLAG HRP (1:1,000, Sigma-Aldrich A8592), mouse anti-MUS81 (1:1,000, Santa Cruz sc-47692), mouse anti-BLM (1:500, Santa Cruz sc-70426), rabbit anti-KAP-1 pSer842 (1:1000, Abcam ab70369), rabbit anti-CHK1 pSer345 (1:1000, Cell Signaling 2341), mouse anti-histone H2A.X pSer139 (1:1000, Millipore 05-636-1), rabbit anti-GEN1 (1:100, raised against GEN1^890–908)19 (link). Uncropped blots are shown in Supplementary Fig. 5.

Cells were incubated in lysis buffer (1× LDS sample buffer (Thermo Fisher Scientific) containing 0.1 M DTT (Amresco), Benzonase (1:3000; Sigma) and protease inhibitors (Roche)) for 20 min at 4°C with shaking, followed by manual scraping. Following SDS-PAGE, proteins were transferred to a nitrocellulose membrane using the iBlot Dry Blotting System (Thermo Fisher Scientific) and blocked for 1 h in TBST containing 5% non-fat milk. Incubation was performed with mouse anti-PLK1 (1:100, clone F8, Santa Cruz Biotechnology) at 4°C overnight and HRP-conjugated donkey anti-mouse IgG (1:3000, BioLegend) for one hour at room temperature. Signal was developed using SuperSignal West Pico chemiluminescent substrate (Thermo Fisher Scientific). Antibodies were then stripped using Restore western blot stripping buffer (Thermo Fisher Scientific) and the membrane was re-probed using mouse anti-β-actin (1:15 000, clone AC15, Thermo Fisher Scientific). Band intensities were quantified using ImageJ.