Cyclin e

Cells were harvested and lysed with ice-cold lysis buffer (50 mM Tris-HCl, pH 7.4, 100 mM 2-Mercaptoethanol, 2% w/v SDS, 10% glycerol). After centrifugation at 10000 × g for 10 min at 4°C, proteins in the supernatants were quantified and separated by 10% SDS PAGE. Western blot assay was performed using anti-WWOX, β-Catenin, Cyclin A, Cyclin D1, Cyclin E, c-myc and p21 antibodies (Abcam, USA). Protein levels were normalized to total GAPDH, using a rabbit anti-GAPDH antibody (Santa Cruz, USA).

To examine the expression of P53, P19, P16^INK4a, P21^Cip1/Waf1, cyclinD1, CDK4, CDK2, and CyclinE, which are markers of cell senescence, in freshly isolated Sca-1⁺ HSPCs from each group, we performed western blot analysis. Cells were lysed in buffer [20 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 1% Triton X-100, and complete protease inhibitor mixture tablets (Beyotime Institute of Biotechnology, Shanghai, China)] and centrifuged at 1,000 g for 5 min at 4°C. Protein concentrations of the cell were determined by a BCA protein assay reagent kit (Beyotime Institute of Biotechnology, Shanghai, China). The cell lysates (40 μg/well) were subjected to SDS-PAGE and transferred onto a PVDF membrane (Millipore, Bedford, MA, USA). The blotted membranes were incubated with primary antibodies to p53 (Abcam, Cambridge, UK), P19 (Abcam, Cambridge, UK), P16^INK4a (ANBO, USA), P21^Cip1/Waf1 (Ptglab, USA), cyclinD1 (ANBO, USA), CDK4 (Ptglab, USA), CDK2 (ANBO, USA), and CyclinE (Abcam, Cambridge, UK) or ACTB (Ptglab, USA). Corresponding horseradish peroxidase-conjugated antibodies were used as the second antibody (ZSGB-BIO, Beijing, China). The membranes were visualized using the enhanced chemiluminescence detection system (Pierce). The level of β-actin was used as an internal control. Relative intensities were quantified using Quantity One (Bio-Rad).