MCF7 Cell Protein Extraction and Western Blot

MCF7 cells were washed once with sterile PBS and harvested using a rubber cell scraper as previously described [34 (link)], with the following changes: cells were pelleted via centrifugation at 1000 rpm and resuspended in ice cold RIPA buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.4, 1 mM EGTA, 1% NP-40) with protease and phosphatase inhibitors. The cell suspensions were then sonicated with a 70% duty pulse sonication cycle, and centrifuged to remove cell debris. The antibodies used in this study, typically at a 1:1000/2000 dilution, included APC1^tot (Abcam133397), APC1^S355phos (Abcam10923), CDC20 (PA5-34775), FZR1/CDH1 (Sigma), CDC27 (Abcam10538), Cyclin B1 (Sigma), HURP (Abcam70744, Proteintech), Securin (Abcam79546), MDR-1 (Sigma), BCRP (Santa Cruz Biotechnology, Dallas, TX, USA; SCBt), TFPI (Abcam, Cambridge, UK), PARP (Sigma), γH2AX (NovusBio, Centennial, CO, USA), histone H3^K9Ac (Millipore, Burlington, MA, USA), histone H3^S10phospho, histone H3^tot (Millipore), GAPDH (Millipore), and tubulin (Sigma). Following primary antibody incubation overnight at 4 °C, the blots were probed with a 1:10,000 dilution of a horseradish peroxidase (HRP) secondary antibody.