Fzr1 cdh1

Sources and catalogue numbers of antibodies were as follows: Cell Signaling Technology: PTEN (#9559), pS473-Akt (#4060) and pSer240/244-S6 (#2215); BD Pharmingen: p27 (#610242), cyclin-D1 (#556470), BrdU (#347580) and Aurora A Kinase (#610939); NeoMarkers: Ki67 (#RM-9106-S); Abcam: NICD (#ab27526), desmin (ab15200) and PTEN (ab32199); Santa Cruz Biotechnology: VE-cadherin (sc-6458), Geminin (#sc-13015), cyclin-A (#sc-53230), Hes1 (#sc-25392) and Erg (sc-353); Millipore: Plk1 (#06-813) and Fzr1/Cdh1 (#CC43); Sigma-Aldrich: β-actin (A5441) and α-tubulin (T6074). Isolectin GS-IB₄ and secondary antibodies conjugated to Alexa 488, Alexa 568 and Alexa 633, and Click-iT EdU Alexa 488 and 647 Imaging Kit were from Molecular Probes. Human (#1506-D4) and mouse Dll4 (#1389-D4) were from R&D Systems. The GDC-0941 compound was from Chem Express Haoyuan (China). The VX680 (MK-0457) compound was from Selleckchem (USA). All chemicals, unless otherwise stated, were from Sigma-Aldrich.

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.