Anti rb

Cells were lysed with SDS lysis buffer (100 mM Tris, pH 8.8, 1% SDS, 5 mM EDTA, 20 mM DTT, and 2 mM AESBF). Total cell extracts were resolved on an SDS polyacrylamide gel using the Mini-PROTEAN Tetra cell System (Bio-Rad) and blotted onto a Hybond P PVDF membrane (GE Healthcare) using the Mini Gel Tank transfer system (Life Technologies). After being blocked with PBST 5% non-fat dry milk (Bio-Rad), membranes were incubated over night with primary antibodies at +4°C, washed and hybridized for 1 h at room temperature using the appropriate horseradish peroxidase-conjugated secondary antibody (rabbit and mouse, Bio-Rad, Hercules, California, USA). Detection was performed with the ECL chemiluminescence kit (Perkin Elmer, Waltham, Massachusetts, USA). The following antibodies were used: anti-FOXM1 (Ab184637, Abcam; dilution 1:300), anti-p63 (Ab735, Abcam; dilution 1:300), anti–β-actin (Sigma; dilution 1:30000), anti-p16 (SC-56330 (JC8), Santa Cruz Biotechnology; dilution 1:1000), anti-Rb (BD554136, BD Biosciences; dilution 1:500), anti-Keratin10 (PRB-159P, Covance; dilution 1:1000), anti-HA (16612, Covance; dilution 1:1000), anti-cMyc (SC-40 (9E10), Santa Cruz Biotechnology; dilution 1:200).

Cells were lysed in RIPA buffer containing Protease Inhibitor Cocktail (Roche). Sample proteins (20 μg) were separated by SDS-PAGE and then transferred to PVDF membranes. After blocking in 10% nonfat milk for 1 h at room temperature, membranes were incubated with primary antibodies overnight at 4°C or 2 h at room temperature. The membranes were then incubated with horseradish peroxidase-labeled secondary antibodies and visualized with ECL. The following primary antibodies were used: anti-p53 (1:500, CST), anti-phosphorylated p53 (Ser15) (1:500, CST), anti-acetylated p53 (1:800, CST), anti-Chk2 (1:500, BD Biosciences), anti-p21 (1:500, BD Biosciences), anti-p27 (1:1800, BD Biosciences), anti-p16 (1:1,200, SAB), anti-p19 (1:600, Thermo), anti-Rb (1:500, BD Biosciences), anti-p130 (1:800, Abcam), anti-Caspase 3 (1:800, CST), anti-PARP (1:800, CST), anti-Bax (1:500, CST), anti-CDK4 (1:800, CST), anti-CDK6 (1:800, CST), anti-E2F1 (1:500, BD Biosciences), anti-Foxo1 (1:500, Millipore), anti-Foxo3a (1:500, Millipore), anti-Sirt1 (1:800, CST), and anti-γ-tubulin (1:8000, Millipore).

Western blot assay was proceeded as standard procedure. The membranes were incubated overnight at 4°C with anti-CDK3, anti-CDK6, anti-E2F2, anti-Rb or anti-p-Rb antibodies (BD PharMingen). Blotted membranes were blotted with an anti-a-Tubulin rabbit monoclonal antibody (Sigma) as a loading control.

Anti-p53, anti-p21^waf1, anti-cyclin D1, and anti-bcl2 antibodies were purchased from Dako (Copenhagen, Denmark). Anti-HNF-1β, anti-GSK-3β, anti-Rb, anti-p27^kip1, anti-XIAP, anti-bax, and anti-integrin β1 antibodies were obtained from BD Biosciences (San Jose, CA, USA). Anti-ARID1A, anti-cyclin B1, and anti-MDM2 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-Snail, anti-Akt, anti-phospho(p) Akt Serine473, anti-pGSK-3β Serine9, anti-pRb Serine807/811, anti-cleaved caspase-3, and anti-integrin β3 antibodies were from Cell Signaling Technology (Danvers, MA, USA). Anti-fibronectin (FN), anti-E-cadherin, and anti-β-actin antibodies were obtained from Abcam (Cambridge, MA, USA), Takara (Shiga, Japan), and Sigma-Aldrich Chemicals (St. Louis, MO, USA), respectively. Anti-cyclin A2 and anti-integrin β2 antibodies were from Novocastra (Newcastle, UK) and Merck KGaA (Darmstadt, Germany), respectively. FN (catalog number #F2006) and cisplatin (CDDP: #479306) were purchased from Sigma-Aldrich Chemicals.