Col3a1

NRCFs or left ventricular tissues were homogenized in lysis buffer containing PMSF (Keygen). To obtain proteins of high purity, lysates were centrifuged at 12,000
g for 15 min. The bicinchoninic acid protein assay kit (Thermo Fisher) was used to measure the protein concentration. Each sample containing 20 μg of protein was separated by 10% SDS-PAGE (Bio-Rad) and then transferred onto a PVDF membrane (Millipore, Billerica, USA). Thereafter, the proteins were probed with primary antibodies against α-SMA (1:1000; Abclonal, Wuhan, China), CTGF (1:500; Abclonal), Col1a1 (1:1000; Abclonal), Col3a1 (1:1000; Abclonal), Smad7 (1:500; Abclonal), Smad3 (1:500; Abclonal), phospho-Smad3 (1:500; Abclonal), and GAPDH (1:2000; Abclonal). Subsequently, the membranes were incubated with a secondary antibody, namely, HRP-linked goat anti-rabbit IgG (1:5000; Abclonal). Finally, the signals were detected using an ECL kit (Tanon, Shanghai, China) on an imaging system (Tanon), and the blots were quantified with ImageJ software.

Cells were washed with 4°C PBS and lysed in lysis buffer (Beyotime) with protease and phosphatase inhibitors (Beyotime). Lysates were centrifuged at 12,500 × g and 4°C for 15 min. Supernatants were collected and proteins were denatured with the NuPAGE^® LDS Sample Buffer (4×) (Invitrogen; Carlsbad, CA, USA). Proteins were separated on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred onto polyvinylidene difluoride (PVDF, Invitrogen) membranes using an iBlot^® Gel Transfer Device (Invitrogen). Membranes were blocked in 5% skimmed milk and then incubated with specific antibodies including hTERT (1:1000; Solarbio), COL1A1 (1:1000; ABclonal; Wuhan China), COL3A1 (1:1000; ABclonal), ELN (1:1000; ABclonal), MMP2 (1:1000; Abcam; Cambridge, UK), MMP3 (1:1000; Abcam), MMP9 (1:1000; Abcam), TIMP1 (1:1000; ABclonal), TIMP2 (1:1000; ABclonal), and GAPDH (1:1000; Abcam). Then, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Solarbio) at room temperature for 2 h. Rapid Step ECL Reagent (Millipore, Schwalbach, Germany) was used to visualize the immunoblot.