Lyses buffer

Cell lysates and tissues were homogenized in lyses buffer (Beyotime, China), and protein concentration was determined by the Bradford assay (Bio-Rad, Hercules, CA). The analysis of protein was performed according to standard SDS-PAGE. After blocking, the membranes were incubated with various specific primary antibodies against β-arrestin2 (3857, 1:800), TLR4 (14358, 1:1000), phospho-IKKβ (2697, 1:1000), IKKβ (8943, 1:1000), phospho-TAK1 (4508, 1:1000), TAK1 (5206, 1:1000), TAB1 (3225, 1:1000), H3 (4499, 1:1000), phospho–p65 (3033, 1:1000), p65 (8242, 1:1000), β-actin (3700, 1:5000) from Cell Signaling Technology, D2R (AB1558, Millipore, 1:1000), and α-synuclein (610786, 1:1000, BD Biosciences) in TBST at 4 °C overnight. After washing, the bands were incubated with corresponding horseradish peroxidase–conjugated secondary antibodies (Santa Cruz Biotechnology, USA) for 1 h at room temperature, and signals were detected by enhanced chemiluminescence (ECL) Western blot detection reagents (Pierce, Rockford, IL). The membranes were scanned and analyzed using ImageQuant™ LAS 4000 imaging system (GE Healthcare, Piscataway, NJ, USA).

The left ventricle was homogenized in lyses buffer (Beyotime, China) and protein concentration was determined by the Bradford assay (Bio-Rad, Hercules, CA, USA). Proteins were separated on 10% Tris-HCl polyacrylamide gels (Bio-Rad) and transferred to a PVDF membrane. After blocking, the blots were incubated with anti-phospho-AMPK (2531, Cell Signaling Technology, Beverly, MA, USA), anti-AMPK (2532, Cell Signaling Technology, Beverly, MA, USA), anti-Kir6.2 (sc-20809, Santa Cruz Biotechnology) and anti-Kir6.1 (sc-11224, Santa Cruz Biotechnology) in TBST overnight at 4°C, and then with horseradish peroxidase (HRP) conjugated secondary antibodies. Immunoreactive bands were detected by enhanced chemiluminescence (ECL) plus detection reagent (Pierce, Rockford, IL, USA), and analyzed using an Omega 16ic Chemiluminescence Imaging System (Ultra-Lum, CA, USA).