Tris glycine mini gel

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 blots were performed using antibodies for anti-FOXM1 (sc-502) and anti-HA (sc-543) and anti-β-actin (ab6276) purchased from Abcam. Fluorescent imaging of GFP-FOXM1 was performed using an anti-GFP antibody (ab290) purchased from Abcam. Cell lysates were prepared using RIPA buffer (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1 % NP-40, 1 % sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM ß-glycerophosphate, 1 mM Na₃VO₄) with proteasome inhibitors (Roche). Lysate was agitated for 30 min at 4 °C by end-to-end rotation, supernatant collected following centrifugation (13,000 rpm, 4 °C, 10 min) and protein concentration measured by BCA (Pierce) assay. Samples loaded onto 4–12 % Tris-Glycine mini gels (Invitrogen), purified by SDS-PAGE and transferred to nitrocellulose membrane (Invitrogen). Membranes were incubated in Odyssey blocking buffer (LiCor) for 1 h at room temperature and probed with FOXM1, HA, or GFP antibody 1:1,000 and β-actin 1:5,000 overnight at 4 °C. For detection, the blot was incubated with LiCor IRDye secondary antibodies; 680LT goat anti-rabbit IgG and 800LT goat anti-mouse IgG both at 1:10,000 and visualized using an Odyssey scanner.

Cells were washed with ice-cold PBS and lysed in RIPA buffer (150 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% sodium deoxycholate, 1% SDS, 2% Octyl-β-d-glucopyranoside, 0.5 mM DTT) containing protease and phosphatase inhibitor cocktails (both from Roche). Samples were lysed on ice for 30 minutes, sonicated, and microcentrifuged at 18,000 RCF for 20 minutes at 4°C. Protein concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Samples were boiled in reducing Laemmli sample buffer, and 10 μg of protein per lane was separated on Tris-Glycine Mini Gels (Invitrogen, Thermo Fisher Scientific). Proteins were transferred onto the PVDF membrane using the Transblot Turbo transfer system (Bio-Rad). Membranes were blocked in 5% nonfat dried milk in 0.1% TBS with Tween (TBS-T) for 1 hour at room temperature and then incubated with primary antibodies overnight at 4°C. After washes with 0.1% TBS-T, membranes were incubated with appropriate HRP-conjugated secondary antibodies (Dako). Finally, the signal was developed with Supersignal West Pico ECL substrate (Thermo Fisher Scientific) or Clarity Western ECL substrate (Bio-Rad) and exposed to Super RX-N FUJIFILM Medical X-ray Film. See complete unedited blots in the supplemental material.