Thermo scientific pierce ecl western blotting detection reagents

Following the treatments, cells were washed twice with cold PBS and lysed in buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM tetrasodium phosphate, 40 mM β-glycerophosphate, 2 mM ethylenediaminetetraacetate (EDTA), 2 mM ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetate (EGTA), 1% Triton X, 10% glycerol, 50 mM sodium fluoride, 0.2 mM sodium orthovanadate and protease inhibitors. The cell lysates were transferred to 1.5-ml Eppendorf tubes, homogenized and centrifuged at 10,000 × g for 5 min. at 4°C. The supernatant was transferred to a fresh tube, and protein concentration was determined using the Bio-Rad Protein Assay Reagent (Bio-Rad). Equal amounts of protein samples were resolved on SDS-PAGE and transferred onto Amersham Hybond ECL 0.45 μM nitrocellulose membranes (GE Healthcare Bio-Sciences, Piscataway, NJ, USA). The membranes were immunoblotted with actin, AMPK, P-AMPKα^Thr172, Bcl-2, caspase 3 full length, eNOS, P-eNOS (Cell Signaling, Danvers, MA, USA), AT1R, AT2R or p53 (Santa Cruz, Dallas, TX, USA) antibodies, followed by secondary antibodies. The signals were visualized using Thermo Scientific Pierce ECL Western Blotting Detection reagents (Thermo Fisher Scientific) at VersaDoc 3000 Gel Imaging System (Bio-Rad).

Protein concentration in homogenate and mitochondria was determined by the Bradford assay (Bio-Rad, Hercules, CA). Equal amounts (50 μg/well) of protein were loaded onto 10% SDS-PAGE gels, run, and transferred onto Amersham Hybond ECL nitrocellulose membranes (GE Healthcare Bio-Sciences Corp., Piscataway, NJ). The membranes were immunoblotted with AMPK, P-AMPKα₁^Thr172 (Cell Signaling, Boston, MA), PPARα, and P-PPARα^Ser21 (Santa Cruz Biotechnology, Santa Cruz, CA). The signals were visualized using Thermo Scientific Pierce ECL Western Blotting Detection reagents (Thermo Scientific, Rockford, IL) at the VersaDoc 3000 Gel Imaging System (Bio-Rad, Hercules, CA).