Ecl western blotting substrate reagent

Cell protein was extracted using a RIPA lysis buffer (Cell Signaling Technology) in the presence of the phosphatase inhibitor cocktail (Thermo Fisher Scientific). Protein concentrations were measured using a BCA Protein Assay Kit (Pierce). Protein samples were separated by SDS-PAGE (4–15%) and electro-transferred onto a PVDF membrane, which was then blocked with 5% BSA for 60 min. The membrane was then incubated with primary Abs overnight at 4°C. After incubation, the membrane was washed 3 times with TBST, incubated with secondary Abs for 60 min at room temperature and developed using the ECL Western blotting substrate reagent (Thermo Fisher Scientific). The signal intensity was analysed by ImageJ and normalized to β-actin.

Total protein was extracted using a RIPA lysis buffer (Cell Signaling Technology) in the presence of a phosphatase inhibitor cocktail (Thermo Fisher Scientific). Protein concentrations were measured using a BCA Protein Assay Kit (Pierce). Protein samples were separated by SDS-PAGE (4–15%) and electro-transferred onto a PVDF membrane (Bio-Rad), which was then blocked with 5% BSA for 60 min. The membrane was incubated with primary Abs overnight at 4°C. After incubation, the membrane was washed 3 times with TBST, incubated with secondary Abs for 60 min at room temperature and developed using the ECL western blotting substrate reagent (Thermo Fisher Scientific). The signal intensity was analyzed by Image J and normalized to β-actin. Hepatic cytokine levels were assayed using specific ELISA kits according to the manufacturer’s instructions. ELISA kits specific to mouse TNF-α, IFN-γ, IL-10, IL-4, and IL-17A were purchased from Biolegend. The IL-1β ELISA kit was purchased from Thermo Fisher Scientific, and the IL-6 ELISA kit was purchased from R&D Systems.