Anti ampk

The assay kits of glucose, triglycerides (TGs), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), superoxide dismutase (SOD), malondialdehyde (MDA), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and Oil Red O staining were obtained from Nanjing Jiancheng Bioengineering Institute, China. Enzyme-linked immuno-sorbent assay (ELISA) kits of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6 were obtained from Lianke Biotechnology, Hangzhou, China. ELISA kit of insulin was purchased from Elabscience Biotechnology, Wuhan, China. The kit for HE staining was purchased from Solarbio, Beijing, China. The BCA assay kit was obtained from Biosharp, China.
Primary antibodies for TRPV1 (1 : 1000) and SOD1 (1 : 200) were from Santa Cruz Biotechnology, USA. Antibodies for SIRT6 (1 : 1000), glucose transporter type 4 (GLUT4, 1 : 1000), SOD2 (1 : 2000), and GAPDH (1 : 20000) were obtained from ProteinTech, China. Antibodies for Nrf2 (1 : 1000), phosphor-IRS-1 (Tyr895) (1 : 1000), and IRS-1 (1 : 1000) were purchased from Cell Signaling Technology, USA. Anti-AMPK (1 : 1000) and anti-phosphor-AMPK (1 : 1000) were purchased from Bioworld Technology, China. Second antibodies for rabbit-source and mouse-source were obtained from Abmart, Shanghai, China.

After the experiments were completed, the C2C12 myotubes, mice skeletal muscle, and liver tissue lysates were prepared using a standard protocol. In brief, the cell and tissue samples were mixed with sample buffer (250 mM tris-hydrochloride (pH 6.8), 0.5 M dithiothreitol (DTT), 10% sodium dodecyl sulfate (SDS), 0.5% bromophenol blue, 50% glycerol, and 5% 2-mercaptoethanol) and denatured at 95°C for 5 min. Sample proteins (50 µg) were separated via 10% SDS-polyacrylamide gel electrophoresis (PAGE) and electrotransferred to nitrocellulose transfer membranes (Whatman, 401396, Dassel, Germany), which were incubated overnight at 4°C with 5% skim milk or 5% bovine serum albumin and a range of antibodies. Anti-AMPK, anti-phospho-(p)-AMPK, anti-AKT, anti-p-AKT, anti-GLUT4 (Bioworld Technology), and anti-β-actin (Bethyl Laboratories) antibodies were the primary antibodies used, and a horseradish peroxidase- (HRP-) conjugated goat anti-rabbit immunoglobulin G (IgG) (Santa Cruz Biotechnology) was used as the secondary antibody. The antigen-antibody reaction was detected using a SuperSignal West Femto maximum sensitivity substrate (Thermo Fisher Scientific, Rockford, IL, USA).