Image systems

Total protein was isolated from heart tissue with a RIPA buffer, and the protein concentration was measured with a BCA assay kit. The protein samples were separated by SDS-polyacrylamide gels with suitable concentration and then transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% bull serum albumin and then developed with diluted antibodies for Bax (14796, 1:1000, Cell Signaling Technology, Beverly, MA, USA), Bcl-2 (BS1511, 1:1000, Bioworld, Bloomington, USA), Cleaved Caspase-3 (9661, 1:1000, Cell Signaling Technology, Beverly, MA, USA), METTL3 (ab240595, 1:1000, Abcam, Cambridge, MA, USA), FTO (27226-1-AP, 1:1000, Proteintech, Wuhan, China), YTHDF1 (17479–1-AP, 1:1000, Proteintech, Wuhan, China), and GAPDH (10494-1-AP, 1:5000, Proteintech, Wuhan, China) overnight at 4 °C. Subsequently, membranes were incubated with horseradish peroxidase- (HRP-) conjugated secondary antibody at room temperature for 90 min. The membranes were observed on Image Systems (Bio-Rad, Hercules, CA, USA). Image Lab software was used for semiquantitative calculation.

Myocardial tissues from left ventricle or peri‐infarcted area were homogenized with RIPA lysis buffer containing PMSF and protease inhibitor. The protein concentration was quantified by using a BCA Protein Assay Kit (PQ003, Shaanxi ZhongHuiHeCai bio‐pharmaceutical Technology Co., Ltd. Xi'an, China), and diluted to a final protein concentration of 5 µg µl⁻¹. Proteins were transferred onto polyvinylidene fluoride (PVDF) membranes and incubated with 5% blocking buffer (Bovine Serum Albumin, BSA) for 2 h at room temperature. The membranes were incubated with the following diluted primary antibodies overnight at 4 °C: tTH (a rate‐limiting enzyme in NE synthesis; Abcam, ab112, 1: 1000), glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) (Genetex, GTX100118, 1: 5000), collagen‐1 (Genetex, GTX26308, 1:1000) or collagen‐3 (Genetex, GTX637655, 1: 500). At the following day, after washing with 1 × Tris‐Buffered Saline Tween‐20 (TBST), the membranes were incubated with the secondary antibodies which were diluted in 5% milk. The protein bands were detected with enhanced chemiluminescence (Bio‐Rad, USA) and the Image Systems (Bio‐Rad, USA).

The total protein was extracted from testicular tissues using cold RIPA buffer. The protein concentration was evaluated by a BCA Protein Assay kit. Equal amounts of protein samples were separated by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% nonfat dry milk for 1.5 h at room temperature and then incubated with the primary antibodies at 4 °C overnight. The antibodies that we used include: 4-HNE (Abcam, Cambridge, MA, USA), Nrf2 (Santa Cruz Biotechnology, Dallas, TX, USA), HO-1 (Abcam, Cambridge, MA, USA), NQO1 (Abcam, Cambridge, MA, USA), Bax (Cell Signaling Technology, Beverly, MA, USA), Bcl-2 (Bioworld, Bloomington, MN, USA), Cleaved Caspase-3 (Cell Signaling Technology, Beverly, MA, USA) and GAPDH (Proteintech, Wuhan, China). The membranes were incubated with the appropriate horseradish-peroxidase (HRP)-conjugated secondary antibodies at room temperature for 1.5 h. Immunopositive bands were visualized by the Image Systems (Bio-Rad, Hercules, CA, USA). The density of immunoblots was calculated by Image Lab software.