Cyclin b

Cultured cells in eight-well chamber slides (Millicell EZ SLIDES) were fixed with cold methanol for 15 min at −20 °C. Then cells were incubated with the following primary antibodies at 4 °C overnight: β-catenin (Cell Signaling, 8480S), ABC (Cell Signaling, 8814S), cyclin A (Sigma, C4710), cyclin B (Sigma, C8831), cyclin D1 (Cell Signaling, 2926), and cyclin E (Cell Signaling, 4129). The cells were then incubated with appropriate fluorochrome-conjugated secondary antibodies as described above. Isotype-matched control antibodies were used as negative controls. Nuclei were counterstained with DAPI Staining Solution, and then images were captured using Olympus inverted fluorescence microscope (IX73). For quantitative analysis of mean fluorescence intensity, cells with positive signals in at least six random fields were measured by Olympus cellSens software.

Whole-cell lysates were prepared from cell lines with RIPA lysis buffer kit (Santa Cruz Biotechnology, Santa Cruz, CA), and the protein concentrations were quantified using a Bio-Rad protein assay (Bio-Rad, Hercules, CA). Whole-cell proteins (30 μg) were separated on 8% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Amersham Corp., Arlington Heights, IL). The membranes were then probed with antibodies against the proteins of H3F3b, BAG-2, Paip2 (Santa Cruz, Santa Cuz, CA), BMI-1 (Millipore, Temecula, CA), EGFR, Bax, and β-actin (Sigma), respectively, for 24 hours. Moreover, membranes were also probed with specific cell cycle, apoptotic and anti-apoptotic antibodies against the following proteins: cyclin A, cyclin B, cyclin D1, p53, caspase 3, caspase 9, NFkb MDM2, BCL2, and BCLxl (Sigma). Washed blots were then incubated with horseradish peroxidase-conjugated anti-mouse antibody (Santa Cruz Biotechnology) for one hour at room temperature. Blots were developed using a peroxidase reaction and visualized with the ECL detection system (Bio-Rad,).

Cell lysates were prepared by incubation with radioimmunoprecipitation assay buffer (Santa Cruz) supplemented with a cocktail of protease inhibitors (Santa Cruz) and the total protein concentrations were determined using bicinchoninic acid method (BioVision). Then 30 µg of proteins were subjected to SDS-polyacrylamide gel electrophoresis before being electroblotted onto a 0.2 μm nitrocellulose membrane (GE Healthcare). After blocking with 5% nonfat dry milk in TBST [25 mmol/l Tris (pH, 7.4), 137 mmol/l NaCl, 0.5% Tween20], membranes were incubated at 4 °C overnight with following primary antibodies: LGR5 (Invitrogen, PA5-35304), ALDH1 (BD Biosciences, 611194), OCT4 (Abcam, ab18976), β-catenin (Cell Signaling, 8480S), ABC (Cell Signaling, 8814S), cyclin A (Sigma, C4710), cyclin B (Sigma, C8831), cyclin D1 (Cell Signaling, 2926) and cyclin E (Cell Signaling, 4129), ZEB1 (Santa Cruz, sc-25388), fibronectin (Sigma, F3648), and E-Cadherin (BD Biosciences, 562869). β-actin (Santa Cruz, sc-47778) was used as loading control. After extensively washing, membranes were incubated with HRP-conjugated secondary antibodies (Santa Cruz) and blot signals were developed with ECL^TM Western Blotting Detect Reagents (GE Health Care).