Image j software version 6

Total proteins of livers in different groups were harvested using RIPA buffer (Sigma-Aldrich, St. Louis, MO, USA) supplemented with a protease/phosphatase inhibitor (Sigma-Aldrich, St. Louis, MO, USA). Protein concentrations were then determined with the BCA protein detection kit (Thermo Fisher Scientific, Beijing, China). A 20 μg sample of protein was separated by 10% SDS-PAGE gels and then electrically transferred onto PVDF membranes (Millipore, Boston, MA, USA). The membranes were then blocked for 2 h with solutions containing 5% of non-fat milk. After washing, the membranes were incubated with a primary antibody (Abcam, Cambridge, MA, USA) overnight at 4 °C and then incubated with a labeled secondary antibody (Boster, Pleasanton, CA, USA) for 1 h at room temperature. Protein bands were visualized via Image J software, version 6.1 (Bio-Rad, Hercules, CA, USA). The GAPDH level was used as a loading control.

Total protein was extracted by lysis buffer for Western blotting with 100 mM of phenylmethanesulfonylfluoride, and 25 μg of total protein sample was subjected to SDS-polyacrylamide gel electrophoresis under reducing conditions. Separated proteins were transferred to nitrocellulose membranes in Tris-glycine buffer containing 20% methanol at 4 °C. The membranes were blocked with 5% skim milk for 2 h, following incubated overnight with diluted primary antibodies against Bcl-xl (1:5000, Abcam, Cambridge, UK), caspase 3 (1:2000, Cell signaling technology, Boston, MA, USA), Bip (1:2000, Cell signaling technology), PERK (1:5000, Abcam), p-PERK (1:1000, Bioss), ATF4 (1:2000, Proteintech, Chicago, IL, USA), ATF6 (1:2000, Proteintech), eIf2α (1:1000, Proteintech, Wuhan, China), p-IRE1(1:1000, Bioss), CHOP (1:1000, Bioss), NF-κB (1:1000, Bioss), IκB (1:2000, Cell signaling technology), ZO-1 (1:1000, Bioss, Beijing, China), Occludin (1:1000, Bioss), Claudin-1 (1:1000, Bioss), and GAPDH (1:1000, Proteintech, China) followed by goat anti-rabbit IgG (H+L; 1:1000, Proteintech, China). The gray values of protein bands were measured by ImageJ software version 6.1 (Bio-Rad Laboratories, Hercules, CA, USA). The cellular protein PERK was used as an internal control for p-PERK, and GAPDH was used as an internal control for other proteins.