Protein Extraction and Western Blot Analysis of Liver Samples

Liver tissues and primary hepatocytes were harvested and lysed in RIPA buffer (#G2002; Servicebio, Wuhan, China) with the protein concentrations measured by the Pierce™ BCA Protein Assay kit (#23225; ThermoFisher Scientific) [33 (link)–35 (link)]. Next, equal amounts of total proteins were separated by SDS-PAGE and transferred onto PVDF membranes that were then blocked with 5% skimmed milk at room temperature for 1 h and probed with indicating primary antibodies at 4°C overnight. The primary antibodies against NRF2 (#ab92946), β-actin (#ab8226), phospho-NF-κB p65 (p-p65, #ab76302), and total-p65 (t-p65, #ab32536) were purchased from Abcam, while anti-p-AMPKα (#50081) and anti-t-AMPKα (#5832) were purchased from Cell Signalling Technology (Beverly, MA, USA). Anti-PDE4D (#12918-1-AP) was purchased from Proteintech (Rosemont, IL, USA). On the second day, the membranes were incubated with the HRP-conjugated secondary antibodies at room temperature for 1 h. Subsequently, the protein bands were visualized by LumiGLO chemiluminescent substrate and quantified using Image Lab software.