Ampkα1

We performed whole cell protein immunoblotting with the following antibodies: β-actin (Santa Cruz Biotechnologies; sc-47778), AMPKα1 (Abcam; ab3759), and proliferating cell nuclear antigen (PCNA; Thermo Fisher Scientific, Waltham, MA, USA; MA5-11358).

A normal diet was purchased from Jiangsu Synergetic Pharmaceutical Bioengineering Co., Ltd. (Nanjing, Jiangsu, China, XTADM001) and consisted of 4% fat, 73.1% carbohydrate, and 14.2% protein. A high-fat diet was purchased from Jiangsu Synergetic Pharmaceutical Bioengineering Co., Ltd. (Nanjing, Jiangsu, China, XTHF60) and consisted of 60% fat, 20% carbohydrate, and 20% protein. Metformin was purchased from Sigma (St. Louis, MO, USA, D150959-5G). Hematoxylin eosin (H&E) and oil red O (ORO) staining kits were purchased from Solarbio (Beijing, China, G1120 and G1261). The rabbit antimouse adiponectin (ADIPOQ), MCP-1, TNF-α, IL-6, and DAB horseradish peroxidase chromogenic kits were purchased from Beyotime (Shanghai, China). The rabbit antimouse adiponectin receptor II (ADIPOR2), phospho-AMPKα1, AMPKα1, SIRT1, phospho-NF-κB p65, NF-κB p65, SREBP1C, FAS, acetyl coenzyme A carboxylase (ACC), PPAR-γ, SCD1, β-actin, and goat antirabbit IgG antibodies were purchased from Abcam (Cambridge, UK).

Western blotting was performed as described previously (9 (link)) using the following primary antibodies: pan-AMPKα (CST no. 2532), p-AMPKα1/2(T172) (CST no. 2535), AMPKα1 (Abcam no. 3759), AMPKα1 antigen (Abcam no. 40461), AMPKα2 (Abcam no. 3760; NBP2-38532; NBP2-38532PEP), AMPKβ1 (CST no. 4178), AMPKβ2 (Novus Biologicals no. 92286), AMPKβ2 antigen (Novus Biologicals no. 92286PEP), AMPKγ1 (Abcam no. 32508), AMPKγ1 antigen (Abcam no. 218345), AMPKγ2 (Novus Biologicals no. 89324), AMPKγ2 antigen (Novus Biologicals no. 89324PEP), AMPKγ3 (Abcam no. 38228), Vdac (CST no. 4661), Tom20 (CST no. 42406), Cox4 (CST no. 4859), Gapdh (CST no. 2118), α-Tubulin (mouse, Abcam no. 7291), Catalase (rabbit, Abcam no. 16731), and Sec61a (CST no. 14867). Proteins were analyzed in comparison to a common protein standard loaded on the gel, which consisted of a whole-tissue lysate mixture of liver, heart, and skeletal muscle.

Cell lysates were prepared in RIPA lysis buffer containing 50 mM Tris (pH 7.5), 150 mM NaCl, 1 % NP-40, 0.5 % deoxycholic acid, 0.1 % SDS, and 5 mM EDTA with freshly prepared protease/phosphatase inhibitors (2 μg/ml aprotinin, 2 μg/ml leupeptin, 1 μg/ml pepstatin A, 1 mM PMSF, 1 mM Na₃VO₄, 5 mM NaF, and 10 mM Na₄P₂O₇). For western blotting of BH3-only Bcl family proteins and caspase-3, a proteasome inhibitor (10 μM MG-132, Sigma) was added to the protein extracts. Thirty micrograms of total protein were electrophoresed on 8 or 15 % polyacrylamide gels and then transferred to PVDF membranes. Anti-phospho-AMPK T183(α-1)/T172(α-2), phospho-AMPK S491(α-2), AMPK α-1, AMPK α-2, Noxa, Puma (Abcam, Cambridge, UK), phospho-AMPK S485(α-1), AMPK α, Bim, caspase-3, cleaved-caspase-3, p-LKB1, or LKB1 (Cell Signaling Technology, Danvers, MA, USA) antibodies were used. Actin (Sigma) was used as the loading control. To visualize the protein bands, enhanced chemiluminescence (iNTRoN Biotechnology, Gyeonggi-do, South Korea) and a bio-imaging system (BIS 303 PC, Philekorea Technology, Daejeon, South Korea) were used. Normalized band intensity was quantified using Image J software.

All antibodies for Western blotting were purchased from Cell Signaling (Beverly, MA) except AMPKα1, β-actin, and TATA (Abcam, Cambridge, MA) and AMPKα2 (Santa Cruz biotechnology, Santa Cruz, CA). A pharmacological AMPK activator (A769662) and the inhibitor of NF-κB (LY303511) were purchased from Tocris Bioscience (Ellisville, MO). The inhibitors for IKK (BMS345541) and NF-κB (SM7368) were purchased from Sigma-Aldrich. LPS (ultrapure LPS, E. coli 0111: B4) was purchased from InvivoGen (San Diego, CA, USA). Recombinant mouse TNF-α protein, anti-mouse TNF-α neutralizing antibody and isotype control IgG were obtained from R&D Systems (Abingdon, UK).