Complete ultra mini edta free easypack

Proteins were extracted from CP (Additional file 6: Table S3) and lysed in 50 µl of lysis buffer (N-PER; Thermo Fischer Scientific) containing protease inhibitor cocktail (cOmplete ULTRA Mini EDTA-free EASYpack; Roche, Basel, Switzerland). Lysates were clarified by centrifugation at 20,000×g at 4 °C for 10 min, and protein concentrations of the resultant supernatants were determined using BCA Protein Assay Kits (Thermo Fischer Scientific). After 10–15 µg of proteins was heated at 70 °C for 10 min in NuPAGE® LDS Sample Buffer (NP0008; Invitrogen) and NuPAGE® Sample Reducing Agent (NP0009; Invitrogen), samples were electrophoresed on 4–12% NuPAGE® Bis-Tris Mini Gel by NuPAGE® MOPS SDS Running Buffer (20×) (NP0001; Invitrogen) Running Buffer (20×), and then transferred to a polyvinylidene fluoride membrane. Primary antibodies are listed in Additional file 6: Table S3. Signals were detected by chemiluminescence using a WesternBreeze kit (WB7103; Invitrogen). Immunoreactive bands were detected using ImageLab version 4.1 software (Bio-Rad Laboratories, Hercules, CA, USA).

Protein expression levels of SIRT3 and MnSOD were determined by Western blot. Protein was extracted from cells and myocardial tissues using a RIPA lysis buffer (Solarbio, Beijing, China) with a protease inhibitor cocktail tablet (cOmplete ULTRA Mini EDTA-free, Easypack, Roche). Protein density of all samples was measured with a BCA Protein Assay Kit (Beyotime, Haimen, Jiangsu, China) according to the manufacturer’s protocol on a microplate reader (TECAN, Männedorf, Switzerland). Antibodies used were as follows: SirT3 (C73E3) rabbit mAb (Cell Signaling Technology, Danvers, Massachusetts, USA), rabbit monoclonal antibody against SOD2 (MnSOD, OriGene, Beijing, China), mouse anti-β-actin monoclonal antibody (ZSGB-BIO), fluorescein-conjugated affinipure goat anti-rabbit IgG (ZSGB-BIO) and fluorescein-conjugated affinipure goat antimouse IgG. CAT protein level was tested with a human CAT ELISA kit (BG, Shanghai, China). For ELISA, cultured cells were collected in phosphate buffered saline and sonicated, while myocardial tissues were ground on ice and proteins were extracted.