Mouse monoclonal anti cyclind3

Total proteins were extracted using a protein extraction kit purchased from Beyotime (Shanghai, China). The extracts were fractionated on 10% sodium dodecyl sulfate-polyacrylamide gels and then transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, United States). To block non-specific binding, membranes were incubated in 5% BSA and then incubated overnight with primary antibodies. The primary antibodies used were as follows: mouse monoclonal anti-PRLR (1:500; ab2773, Abcam, Cambridge, United Kingdom), mouse monoclonal anti-cyclinD3 (1:200; 2936 s, Cell Signaling Technology, Boston, MA, United States), mouse monoclonal anti-PCNA (1:500; 2586 s; Cell Signaling Technology, Boston, MA, United States), rabbit polyclonal anti-Bax (1:300; 2772 s; Cell Signaling Technology, Boston, MA, United States), and mouse monoclonal anti-Bcl-2 (1:300; 3498 s; Cell Signaling Technology, Boston, MA, United States). After incubating with secondary antibodies, antibody binding was quantified using an ECL reagent (Millipore, Darmstadt, Germany), and bands were visualized using a ChemiDoc™ XRS + (Bio-Rad).

The 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), crystal violet, propidium iodide (PI), RNAse, cell lysis buffer, dimethyl sulfoxide (DMSO), phenylmethylsulfonyl fluoride (PMSF), protease and phosphatase inhibitor cocktails, and trypan blue were purchased from Sigma-Aldrich (St. Louis, MO, USA). The mouse monoclonal anti-PARP antibody, anti-phospho-histone H2A.X (Ser139), mouse monoclonal anti-cdc2 (CDK1) antibody, anti-phospho-cdc2 (Thr161) antibody, and mouse monoclonal anti-cyclin D3 were acquired from Cell Signaling Technology, Inc. (Beverly, MA, USA). The monoclonal anti-β-actin antibody was acquired from Sigma-Aldrich (St. Louis, MO, USA). The monoclonal anti-cyclin B1, goat anti-mouse IgG-horseradish peroxidase (HRP), and goat anti-rabbit IgG-HRP antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).
The phycoerythrin (PE)-conjugated anti-CD133/2 (293C3) antibody was purchased from Miltenyi Biotec (Bologna, Italy). The fluorescein isothiocyanate (FITC)-conjugated anti-CD326 (EpCAM) antibody was purchased from BD (BD, Becton-Dickinson Biosciences, San Jose, CA, USA). The Live/Dead Fixable Far Red Dead cell stain kit (Reactive Dye) was acquired from Thermo Fisher Scientific (Waltham, MA, USA).