Phospho lkb1

Liver tissue was homogenized using Tris-HCl buffer (pH 7.5) containing 150 mM NaCl, 0.1% sodium dodecyl sulfate (SDS), 1% NP-40, and 1% phenylmethylsulfonyl fluoride (PMSF). The tissue extracts were centrifuged at 12,000 ×g at 4°C for 20 min and the protein concentration of the supernatant was determined using a BioRad protein assay according to the manufacturer's protocol (Bio-Rad Laboratories, CA, USA). Equivalent amounts of protein of each sample were separated by 12% SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Millipore, Shanghai, P.R. China). The membranes were then blocked with 5% skim milk and subsequently incubated with the appropriate primary antibodies liver kinase B (LKB1) (Cat No. 3047), phospho-LKB1 (Cat No. 3482), AMPK (Cat No. 2532), phospho-AMPK (Cat No. 2531), acetyl-CoA carboxylase1 (Cat No. 3676), and phospho-acetyl-CoA carboxylase1 (Cat No. 3661) (all from Cell Signaling Technology, Inc., Danvers, MA, USA), and β-actin (Cat No. SC-47778, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The membranes were subsequently reacted with horseradish peroxidase-conjugated secondary antibody (Cat No. 7074S). The immunoreactive bands were visualized by incubation with lumiGLO reagent (Cell Signaling, Beverly, MA, USA) and analyzed using an LAS 4000 chemiluminescent image analyzer (Fuji, Tokyo, Japan).

Mouse kidneys were homogenized in lysis buffer containing RIPA buffer and protease inhibitor (#P8340; Sigma Aldrich). Protein concentration was measured using Protein assay dye reagent concentrate (#5000006, Bio-Rad) which is based on the Bradford method. The protein samples were loaded on 4%–20% gradient pre-cast gels (#4568096; Bio-Rad) and transferred to PVDF membranes. The membrane was blocked with 5% BSA, followed by incubation/blotting with primary antibodies of S6 (#2217L; Cell Signaling Technology, Inc.), phospho-S6 (#5364S; Cell Signaling Technology, Inc.), AMPK alpha (#5831S; Cell Signaling Technology, Inc.), phospho-AMPK alpha (#2535S; Cell Signaling Technology, Inc.), LKB1 (#3047S; Cell Signaling Technology, Inc.), phospho-LKB1 (#3482S; Cell Signaling Technology, Inc.), SIRT1 (#9475T; Cell Signaling Technology, Inc.), LC3-I/II (#4108s; Cell Signaling Technology, Inc.), SQSTM1/p62 (#5114; Cell Signaling Technology, Inc.), and GAPDH (#sc-25778; Santa Cruz Biotechnology, Inc.). The working dilution for all the antibodies was 1:1,000. Goat anti-rabbit HRP conjugated secondary antibody was used against the above primary antibodies (#7074S; Cell Signaling Technology, Inc.). The membranes were exposed to ECL reagent (#NEL104001EA; PerkinElmer) and developed using an X-ray film developer. Band density of each blot was quantified using ImageJ software.