Ecl plus chemiluminescence reagent kit

Mouse myocardial tissue was lysed in a RIPA lysate containing protease inhibitor (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and homogenized on ice. Supernatant was collected following centrifugation at 4,000 × g at 4°C for 10 min to determine protein concentration using the Bicinchoninic acid Protein Assay kit. Protein samples (30 µg) were subjected to 10% SDS-PAGE and transferred to PVDF membranes. Following addition of TGF-β1 (1:1,000; cat. no. ab92486), Smad3 (1:1,000; cat. no. ab40854), phosphorylated (p)-Smad3 (1:1,000; cat. no. ab52903), collagen I (1:1,000; cat. no. ab34710) and collagen III (1:1,000; cat. no. ab7778), Bcl-2 (1:1,000; cat. no. ab59348), apoptosis regulator BAX (Bax; 1:1,000; cat. no. ab32503), caspase-3 (1:1,000; cat. no. ab13847) and GAPDH (1:2,000; cat no. ab8245) antibodies (all Abcam), protein samples were incubated at 4°C overnight. Following addition of goat anti-rabbit immunoglobulin G/horseradish peroxidase conjugated antibody (1:2,000; cat. no. bs-0295G-HRP; BIOSS, Beijing, China), protein samples were incubated at room temperature for 2 hand western blots were developed using ECL-Plus chemiluminescence reagent kit (Thermo Fisher Scientific, Inc.) and visualized by UVP Bio-Imaging Systems. Thereafter, the optical density analysis was performed (Image J 1.8.0 software; National Institutes of Health, Bethesda, MD, USA).

The lysates of brain tissue or cell lysates in different groups were submitted to 10% SDS-PAGE gels for separation, and then were transferred to nitrocellulose membranes. Membranes were blocked with 5% skim milk, and then incubated overnight with the primary antibodies at 4°C. After the membranes were rinsed with TBST, the membrane was incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies for 1 h. The blots were imaged using the ECL Plus chemiluminescence reagent kit (Thermo, USA). The density of the bands was calculated using Image Quant_LAS500 (GE Healthcare).