Phosphorylated p65

Cells were cultured and grown to around 80% confluence, then lysed with PRO-PREP Protein Extraction Solution (iNtRON Biotechnology, Inc. Gyeonggi-do, South Korea) or PARIS™ Kit. All protein concentrations were detected using a BCA Protein Assay Kit (Beyotime, Shanghai, China), and 30 μg of each sample was loaded. After resolved proteins were blotted onto PVDF transfer membranes and blocked with 10% non-fat milk in TBST for 2 h, the membranes were incubated with primary antibodies against ZO-1 (Bioss, Beijing, China), Occludin (Bioss), Claudin-1 (Bioss), p65 (Santa Cruz, Dallas, TX, USA), phosphorylated p65 (Bioss), GAPDH (Bioss) at 4°C overnight. After washed with PBS subsequently, the membranes were incubated with their corresponding secondary antibodies (Beyotime). In addition, 5% BSA (Beyotime) was used to block the membrane in phosphorylated p65 detection.

Proteins from tissues or cells were harvested using radioimmunoprecipitation buffer containing Tris‐HCl (50 mmol L⁻¹), NaCl (150 mmol L⁻¹), MgCl₂ (5 mmol L⁻¹), EDTA (2 mmol L⁻¹), NaF (1 mmol L⁻¹), 1% NP40, and 0.1% SDS. The protein concentrations were quantified using commercial kits (Pierce). All protein samples were equally subjected to 10% SDS polyacrylamide gels, transferred to polyvinylidene difluoride membranes by electrophoresis, incubated with primary and secondary antibodies, and finally visualized by a chemiluminescence detection kit (Millipore). The following primary antibodies were purchased: NR2F6 (Abcam, Catalog number: ab137496; R&D Company, Catalog number: PP‐N2025‐00), CD36 (Abcam, Catalog number: ab133625), GAPDH (Abcam, Catalog number: ab181602), phosphorylated P65 (Beyotime, Catalog number: AF5881), total P65 (Beyotime, Catalog number: AF0246), a‐Tubulin (Sigma‐Aldrich, Catalog number: T6199).