P ampk α1

Total protein was extracted from RAW264.7 macrophages and primary peritoneal macrophages using radio immunoprecipitation lysis buffer (RIPA, Solarbio, Beijing, China) supplemented with phenylmethylsulfonyl fluoride (PMSF) and phosphatase inhibitors (Solarbio, Beijing, China). The protein was collected, and its concentration was measured using a Pierce BCA protein assay kit (Thermo, Rockford, IL, USA). Equal amounts of proteins were separated by electrophoresis on SDS-PAGE gels and subsequently transferred onto PVDF membranes. Blocking was with TBST containing non-fat milk powder, the membranes were incubated overnight at 4 °C with primary antibodies. The primary antibodies against NRF2, p-AKT, AKT, iNOS (Cell Signaling Technology, Danvers, MA, USA), and HO-1, NQO-1, COX-2, p-GSK-3β, GSK-3β, p-AMPK-α1, AMPK-α1, p-ERK, EKR, p-JNK, JNK, p-P38, P38, p-NF-κB p65, NF-κB p65, and GAPDH (ABclonal, Wuhan, China) were diluted to a ratio of 1:2000. Before adding secondary antibodies (goat anti-rabbit or goat anti-mouse) diluted to a concentration of 1:10,000 (ABclonal, Wuhan, China), the membranes were washed four times with TBST for 15 min each time. Membranes were visualized using an Amersham Imager 600 (a gel imaging system from GE Co., Fairfield, CT, USA) after applying enhanced chemiluminescence. For detailed information of WB procedures, refer to our previous study [50 (link)].

The detailed experimental procedures can be found in our previous study [35 (link)]. Antibodies used included p-AKT, AKT, NRF2, and iNOS from Cell Signaling Technology (Danvers, MA, USA) and NQO-1, HO-1, COX-2, p-AMPK-α1, AMPK-α1, p-GSK-3β, GSK-3β, β-actin, and goat anti-rabbit or goat anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibody from ABclonal (Wuhan, China).