Mdr 1

Adherent MCF7 cells were scraped, and centrifuged with sterile PBS for collection and resuspended in RIPA buffer followed by pulse sonication. Westerns were performed as described [14 (link)]. Antibodies against the following proteins were used, typically at 1:2000 dilution: MDR-1 (Sigma), BCRP (Santa Cruz Biotechnology; SCBt), HIF1α (Abcam), S6K total (SCBt), S6K^S411phos (SCBt), p53 (SCBt), p53^S392phos (Abcam), TFPI1 (Abcam), AMPKα1/2 total (SCBt), AMPKα1^T183/2^T172phos (Abcam), AKT total (SCBt), AKT^S473phos (SCBt), PARP (Sigma), ERα (SCBt), histone H3 total (Millipore), H3^K9Ac (Millipore), H2B total (Abcam), H4^K12Ac (Abcam), NFκB (SCBt), NREL (SCBt), tubulin (Sigma), actin (Sigma), and GAPDH (Millipore). Luminescence was captured on film (Kodak) with subsequent chemical development. Collection and semiquantitation of Western blots densitometry was done using ImageJ Version 1.51 from scans of the original film.

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.