Western blot analysis for cyclin D1 and cyclin E1

Protein was extracted from the liver tissue using RIPA Lysis Buffer (Beyotime Institute of Biotechnology, Haimen, China). After centrifugation (15,000 × g, 4°C, 20 minutes), the supernatants of the homogenate were collected for protein concentration measurement. After separation by SDS-PAGE gels, the denatured protein was transferred to nitrocellulose membranes. The membranes were then incubated with primary antibodies to cyclin D1 (1:10000; Abcam), cyclin E1 (1:500; Abcam), and glyceraldehyde 3-phosphate dehydrogenase (1:1000; Abcam). The membranes were then incubated with secondary antibodies followed by use of an enhanced chemiluminescence kit (Pierce Biotechnology, Rockford, IL, USA).