Horseradish peroxidase linked anti mouse or anti rabbit conjugates

The cell lysates were prepared using a RIPA buffer (Thermo Scientific, St. Peters, MO, USA) with a protease inhibitors mixture (Roche Applied Science, Mannheim, Germany). Lysate was agitated for 30 min at 4 °C then centrifuged at 14,000 rpm for 10 min at 4 °C to collect the supernatants, and then the protein concentration was measured using the Bio-Rad DC Protein Assay (Biorad, UK). Then, 20 µg of each protein sample was then loaded onto 4–12% Tris-Glycine mini gels (Invitrogen), and was transferred to nitrocellulose membrane (GE Healthcare, Amersham, Buckinghamshire, UK). The membranes were incubated with the indicated antibodies, and the FOXM1 (C-20) (Cat#sc-502) antibody was purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Other antibodies were purchased from Cell Signalling Technology (New England Biolabs Ltd., Hitchin, UK). Primary antibodies were detected using horseradish peroxidase-linked anti-mouse or anti-rabbit conjugates, as appropriate (Dako, Glostrup, Denmark), and were visualized using the UVITEC chemiluminescence imaging platform (UVITEC, UK). The intensities of the protein bands were then quantitated using an ImageJ program and were normalized by dividing the intensity of the bands with the that of the actin band as the control.

Western blotting was performed on whole-cell extracts by lysing cells in buffer as previously described.^{11 (link)} The antibodies against FOXM1 (C-20)(Cat#sc-502), β-tubulin (H-235) (Cat# sc-9104) and Cyclin B1 (Cat# sc-752) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The KIF20A antibody (ab104118) was purchased from Abcam (Cambridge, UK). The PARP (#9542) and Caspase7 (#9491) antibodies were purchased from Cell Signaling Technology (New England Biolabs Ltd., Hitchin, UK). Primary antibodies were detected using horseradish peroxidase-linked anti-mouse or anti-rabbit conjugates as appropriate (Dako, Glostrup, Denmark) and visualized using the ECL detection system (Amersham Biosciences, Pollards Wood, UK).