Protein lysis kit

Protein extracts were prepared using a protein lysis kit (Beyotime, China) with a protease inhibitor (Beyotime), and protein lysates were separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Next, the proteins in the gel were transferred to polyvinylidene fluoride (PVDF) membranes (Merck Millipore). The membranes were washed and blocked with 5% BSA in Tris-buffered saline and Tween-20 (TBST) for 1 h at room temperature. The membranes were then incubated with primary antibodies, including anti-NF-κB (1: 2000, Proteintech, 66535-1-lg), anti-TLR4 (3.5 μg/mL, Abcam, ab13867), anti-CPT-1 (1: 1000, Proteintech,15184-1-AP), anti-PPAR-α (1 μg/mL, Abcam, ab126285), anti-Claudin1 (1: 5000, Abcam, ab180158), anti-occludin (1: 1000, Abcam, ab167161), and anti-GAPDH (1: 25000, Proteintech, 60004-1-lg) for 16 h at 4°C. After washing with TBST, the membranes were incubated with IgG conjugated with horseradish peroxidase (HRP; Thermo Fisher, USA) for 1 h at room temperature. Finally, enhanced chemiluminescence (ECL; Beyotime) was employed to detect protein bands using the ChemiDoc XRS system (Bio-Rad Laboratories, CA, United States). Densitometry analysis of each band was conducted using ImageJ software.