Camkkβ

Total protein was extracted using RIPA lysis buffer (CWbiotech, Beijing, China) containing 1 mM phenyl methyl sulfonyl fluoride (PMSF; Beyotime, Shanghai, China). The protein concentration was measured using BCA Protein Assay kit (Beyotime). For western blot analysis, equal amounts of soluble protein (50 μg per well) were subjected to 12% SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 5% skimmed milk at 37°C for 1 h and then incubated with the primary anti-LC3, TAK1, LKB1, CaMKK β, AMPK, p-AMPK, mTOR, and p-mTOR antibodies (Abcam, Cambridge, UK) on the rocking bed overnight at 4°C. After three times washe with TBST, the PVDF membranes were incubated with horseradish peroxidase-labeled goat anti-mouse IgG monoclonal antibody or goat anti-rabbit IgG monoclonal antibody in the blocking solution (1:10,000) at room temperature for 1 h. After being washed with TBST, membranes were detected using a DBA kit (Pulilai, Beijing, China). The images were analyzed using the gel analysis software (Quantity one; Bio-Rad, USA). All experiments were repeated three times.

Western blot was performed to detect protein expression as previously described (Yuan et al. 2012 (link)). Briefly, total proteins from primary rat cortical neurons were isolated using the protein buffer (Beyotime, Shanghai, China). Proteins were then separated by 10% SDS-PAGE and transferred onto PVDF membranes. After being blocked with 5% fat-free milk, the membranes were incubated at 4 °C overnight with primary antibodies against p62 (1:1,000, Abcam, Cambridge, MA, USA), Beclin-1 (1:1,000, Abcam), microtubule-associated protein 1 light chain 3 (LC3-I and LC3-II, both from Anti-LC3B antibodies, 1:1,000, Sigma-Aldrich), CaMKKβ (1:1,000, Abcam), AMPK (1:1,000, Cell Signaling Technology Inc., Danvers, MA, USA), phospho (p)-AMPK (1:1,000, Cell Signaling Technology Inc.), mTOR (1:1,000, Cell Signaling Technology Inc.), and p-mTOR (1:1,000, Cell Signaling Technology Inc.). Subsequently, membranes were incubated for 2 h at room temperature with horseradish peroxidase (HRP)-secondary antibodies in TBST. The protein was visualized using the ECL detection system (Beyotime, Shanghai, China). The band intensity was quantified with Image-Pro Plus 6.0 software.

Triglyceride (TG), total cholesterol (TC), and low-density lipoprotein-cholesterol (LDL-c) were from Roche Life Science (Basel, Switzerland). 2-Deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG), STO-609 (CaMKKβ inhibitor), compound C (AMPK inhibitor), and DT-061 (PP2A activator) were from Sigma-Aldrich (St. Louis, United States), and PP2A, AMPK, p-AMPK, CaMKKβ, p- CaMKKβ, and GAPDH were purchased from Abcam (Cambridge, UK).