Anti leptin

The H9c2 cardiac cells were seeded at a concentration of 1 × 10⁴ cells/mL, and incubated at 37°C. The cells with indicated treatments were harvested and dissolved into lysis solution for carrying out western blot testing. Protein concentration was assessed by the bicinchoninic acid protein assay kit. The same amount of cell protein was loaded onto 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis for electroblotting, and then transferred onto a polyvinylidene fluoride membrane. After being blocked in 5% milk without fat, the membranes were incubated with anti‐p38 (1:1,000 dilution; Cell Signaling Technology, Massachusetts, Boston, USA), anti‐p‐p38 (1:1,000 dilution; Cell Signaling Technology), anti‐ERK1/2 (1:1,000 dilution; Cell Signaling Technology), anti‐p‐ERK1/2 (1:1,000 dilution; Cell Signaling Technology), anti‐p‐JNK, anti‐JNK antibody (1:1,000 dilution; Cell Signaling Technology) or anti‐leptin (1:1,000 dilution; Cell Signaling Technology) at 4°C overnight on a shaker. Then the horseradish peroxidase‐conjugated goat anti‐rabbit as a secondary antibody was used at room temperature for 2 h. The membranes were washed three times with phosphate‐buffered saline with Tween 20, and fluorescence was detected by enhanced chemiluminescence (Bio‐Rad Laboratories Inc., Hercules, California, USA) and exposed to X‐ray film.

NRG, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (JC-1), dichlorofluorescein diacetate (DCFH-DH) and Hoechst 33258 were obtained from Sigma-Aldrich (St Louis, MO). Leptin antagonist (LA), leptin receptor antagonist (ObRa), and AG490 were purchased from ProSpec (East Brunswick, NJ). A Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies (Kumamoto, Japan). Fetal bovine serum (FBS) and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Gibco BRL (Grand Island, NY). Anti-STAT3, anti-phospho-STAT3, anti-JAK2, anti-phospho-JAK2, anti-leptin, and anti-leptin receptor antibodies were supplied by Cell Signaling Technology (Boston, MA), and horseradish peroxidase (HRP)-conjugated secondary antibody and bicinchoninic acid (BCA) protein assay kits were obtained from the KangChen Bio-tech, Inc. (Shanghai, China). Enhanced chemiluminescence (ECL) solution was purchased from the KeyGen Biotech (Nanjing, China).