Ab80039

Proteins were extracted from cells or tissues in RIPA lysis buffer supplemented with PMSF and phosphatase inhibitors and dissolved in SDS loading buffer. Equal amounts of proteins were resolved by SDS-PAGE and transferred to a nitrocellulose membrane (Millipore). After being blocked with 5% skimmed milk in 0.25% TBS-Tween (TBST), membranes were incubated with the following primary antibodies overnight at 4°C: p62 (1:2000, Abcam, ab56416), ATG5 (1:2000, Proteintech, No. 10181-2-AP), Beclin-1 (1:2000, Proteintech, No. 11306-1-AP), LC3 (1:2000, Proteintech, No. 14600-1-AP), TET2 (1:1,000, Proteintech, No. 21207-1-AP), p-AMPK (1:2000, Abcam, ab23875), AMPK (1:2000, Abcam, ab80039), SGLT2 (1:1,000, Proteintech, No. 24654-1-AP), SREBP-1c (1:1,000, Abcam, ab28481), PPARα (1:800, Proteintech, No. 15540-1-AP), CD36 (1:1,000, Abcam, No. 18836-1-AP), and β-actin (1:5,000, Bioworld, BS6007M). After washing with TBST, the horseradish peroxidase (HRP)-conjugated secondary antibody was incubated for 1 h at room temperature. Finally, protein bands were visualized with an ECL kit (Advansta, K-12045-D50). Quantification of each band was analyzed with ImageJ software. Proteins levels were normalized against the loading control β-actin.

Western blotting was conducted as previously described [31 (link)]. Proteins from brain samples and cultured BV2 cells were lysed using RIPA lysis buffer. Equal amounts of protein (40 μg/10 μL) were loaded onto sodium dodecyl sulfate–polyacrylamide gels. The proteins were electrophoresed until sufficiently separated and then transferred to PVDF membranes. The membranes were incubated overnight at 4 °C with primary antibodies against rabbit anti-STING (1:1000, Proteintech, Cat.No.19851-1-AP), rabbit anti-TBK1 (1:1000, Proteintech, Cat.No. 28397-1-AP), rabbit anti-TBK1 (phospho S172) (1:2000, Abcam, ab-109272), rabbit anti-AMPK (phospho T183 and T172) (1:1000, Abcam, ab-23875), rabbit anti-AMPK (1:1000, Abcam, ab-80039), rabbit anti- iNOS (1:500, Proteintech, Cat.No. 18985-1-AP), rabbit anti-IL-1β (1:1000, Abcam, ab-9722), rabbit anti-NLRP3(1:1000, Abcam, ab210491), mouse anti-ASC (1:2000, Santa Cruz, sc-271054), goat anti-caspase-1 (1:2000, Santa Cruz, sc-22165), and mouse anti-β-actin (1:5000, Proteintech, Cat.No. 60008-1-Ig). The membranes were processed with horseradish-peroxidase-conjugated secondary antibodies at room temperature for 1 h. Bands were visualized using the ECL Plus chemiluminescence reagent kit (Amersham Bioscience, Arlington Heights, IL). The band densities were quantified with the Image J software (NIH).