Hrp goat anti rabbit igg h l antibody

MAECs were cultured on composite scaffolds for 3 days, and then the total protein of MAECs was extracted by adding radioimmunoprecipitation (RIPA) lysis buffer and protease inhibitor (Beyotime Biotechnology, Shanghai, China) cocktail to plates in an ice bath. The lysates were centrifuged for 10 min with a speed of 12,000 rpm at 4 °C. Protein concentrations of the collected supernatants were determined using a BCA assay (Thermo Fisher, USA). The protein samples were separated by electrophoresis using 10% SDS polyacrylamide gels after being boiled with SDS-PAGE loading buffers (Abclonal Technology Co., Ltd., China) in a hot water bath. The proteins were then transferred to nitrocellulose membranes and treated with primary antibodies overnight at 4 °C. The primary antibodies used for western blots are presented in Additional file 1: Table S4. The membranes were subsequently treated with HRP goat anti-rabbit IgG (H+L) antibody and HRP goat anti-mouse IgG (H+L) antibody (1:2000; Abclonal Technology Co., Ltd., China) and detected by ECL reagent (Abclonal Technology Co., Ltd., China).

Total protein was extracted from the left ventricular myocardium or HL-1 cell lines and lysed via RIPA buffer. Degenerated protein concentration was measured by bichinchoninic acid (BCA) Protein Assay Kit (Beyotime, Shanghai, China). In each experiment, 20 μg proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 12% or 10% polyacrylamide gels) and transferred to nitrocellulose filter membrane. The membrane was blocked for 2 h with 5% skim milk in PBS at room temperature and then incubated at 4°C overnight with primary antibodies Gpx4 (1:500 dilution, A11243, Abclonal, Wuhan, China), Alox15 (1:1000 dilution, A6864, Abclonal, Wuhan, China), GAPDH (1:2000 dilution, A19056, Abclonal, Wuhan, China), and Ptgs2 (1:1000 dilution, A1253, Abclonal, Wuhan, China). HRP Goat Anti-Rabbit IgG (H + L) antibody (1:5000 dilution, AS014, Abclonal, Wuhan, China) was used as the secondary antibody for 1 h at room temperature. The images were captured by Odyssey v3.0 software. Protein bands were measured by the Image J software.