Anti phospho ampkα thr172

Tissues and cells were lysed, and identical amounts of proteins (20 μg/lane) were separated by SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were incubated overnight with the following primary antibodies: anti-NS5A, anti-SREBP-1c (Abcam, Cambridge, UK), anti-AMPKα1, anti-AMPKα2, anti-FASN, anti-ACC1 (Proteintech, Wuhan, China), anti-Flag (Sigma-Aldrich, St. Louis, USA), anti-phospho-AMPKα (Thr172) (Affinity, OH, USA), or anti-β-Actin (RayBiotech, Beijing, China). Detection was performed using the corresponding horseradish peroxide-labeled IgG (ZSJQ-Bio, Beijing, China) followed by chemiluminescence (Advansta, CA, USA).

The expression levels of NS5A, SREBP-1 and phospho-AMPKα (Thr172) in liver samples were measured using IHC staining. In brief, specimens were fixed in 4% paraformaldehyde overnight and then embedded in paraffin wax according to standard methods. Following antigen retrieval by heating the slices in a microwave for 30 min, the deparaffinized liver sections were incubated with a 3% H₂O₂ solution for 30 min to quench endogenous peroxidase activity. The slides were incubated overnight at 4 °C with anti-phospho-AMPKα (Thr172) (Affinity, OH, USA), anti-NS5A or anti-SREBP-1 (Abcam, Cambridge, UK). Negative controls were obtained by omitting the primary antibody and using primary antibody diluent. After washing, the slides were incubated with anti-rabbit or mouse Plus-HRP (ZSJQ-BIO, Beijing, China) for 1 h at room temperature. Then, the sections were incubated with N, N-dimethylaminoazobenzene (DAB) (ZSJQ-BIO, Beijing, China) and the nuclei were counterstained with hematoxylin (ZSJQ-BIO, Beijing, China). The images were captured using an Olympus BX53 microscope at × 400 or × 1000 magnification.