Anti phospho ampkα thr172 antibody

BV2 microglia was washed by cold PBS and lysed in RIPA buffer (Millipore, Temecula, CA, United States) containing Complete Protease Inhibitor Cocktail (Roche Diagnostics, Mannheim, Germany) and 1 mM PMSF. BCA Protein Assay Kit (Pierce Biotechonology, Rockford, IL, United States) was used to determine the concentrations of the protein samples. Thirty μg of total protein was electrophoresed through a 10% SDS-PAGE gel, and then transferred to 0.45 μm nitrocellulose membranes. The membranes were incubated with 5% (v/v) BSA for 2 h. The blots were incubated overnight at 4°C with rabbit-derived anti-AMPKα antibody (2532, Cell Signaling Technology, Danvers, MA, United States), anti-phospho-AMPKα (Thr172) antibody (2535, Cell Signaling Technology), anti-phospho-Acetyl-CoA Carboxylase (Ser79) antibody (11818, Cell Signaling Technology), anti-acetyl-CoA Carboxylase antibody (3676, Cell Signaling Technology) or goat-derived anti-actin antibody (sc58673, Santa Cruz Biotechnology, Santa Cruz, CA, United States), then incubated with HRP-conjugated secondary antibody (Epitomics, Hangzhou, China). Protein signals were visualized using the ECL detection reagent (Pierce Biotechnology, Rockford, IL, United States). The intensities of the bands were analyzed by densitometry using computer-based Image-Pro Plus program.

Protein concentrations of cell lysates for western blot analysis were determined using the Bio-Rad DC protein assay. Equal amounts of protein were separated by SDS PAGE, transferred to nitrocellulose membranes and detected using anti-OTUB1 antibody (Cell Signaling), anti-β-actin antibody (Sigma), anti-AMPKα antibody (Cell Signaling), anti-phospho-AMPKα (Thr172) antibody (Cell Signaling), anti-FIH antibody (Abcam), anti-α-tubulin antibody (Santa Cruz), anti-FLAG antibody (Sigma), anti-V5 antibody (Invitrogen), or anti-HA antibody (Roche).