Complete mini easy pack

Liver tissue was homogenized in RIPA lysis buffer containing protease inhibitor (Complete mini EASY pack, Roche, Basel, Swiss) to extract protein. The concentration was determined by BCA kit (CoWin Bioscience, Beijing, China) and protein was resolved with 10% polyacrylamide gel, then transferred to the PVDF membrane (Millipore, Billerica, MA, USA). After blocking, they were incubated overnight in antibody dilutions of GRP78 (1:1000) (CST, Boston, MA, USA, 3183), P-PERK (1:1000) (CST, Boston, MA, USA, 3179), P-EIF2α (1:1000) (CST, Boston, MA, USA, 3398), P-NFκB (1:500) (CST, Boston, MA, USA, 3033), P-IREα (Abcam, Cambridge, UK, ab148187), TRAF2 (1:1000) (Abcam, Cambridge, UK, ab126758) or β-actin (1:1000) (Huaan biological technology, Hangzhou, China), and secondary antibody were incubated for 2 h at room temperature. The images of blots were acquired by GBOX Chemi XT4 gel imaging system (Syngene, Cambridge, UK).

Liver tissues or cells were homogenized in RIPA buffer with the proteinase inhibitor (complete mini EASY pack, Roche, Basel, Switzerland) and protein concentration was determined through the BCA method. The lysates were resolved on 12% sodium dodecyl sulfate-polyacrylamide by gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). After blocking, membranes were immunoblotted with the antibody against ARPP-19 (Proteintech, Wuhan, China), cyclin A2, cyclin D1, Cdk2, Cdk4, cyclin B1, phospho-cdc2, phospho-(Ser) CDKs substrate (Cell Signaling Technology, Boston, MA, USA) or β-actin (Huaan Biological Technology, Hangzhou, China) as the internal loading control. The primary antibodies were visualized with goat anti-rabbit (CST) peroxidase-conjugated antibody by an enhanced chemiluminescence detection system (Millipore). The images of blots were acquired by the GBOX Chemi XT4 System (Syngene, Cambridge, UK) and quantified by densitometry with GeneTools software (Syngene).