Bc3710

Protein was extracted from the heart tissues and cells using a protein extraction kit (BC3710; Beijing Solarbio Science & Technology Co., Ltd Solarbio, Beijing, China) and protein concentration was quantified using a BCA assay (Thermo Fisher Scientific, Inc.) according to the manufacturer’s protocol. Protein (20 µg) was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane (EMD Millipore). Following blocking with 5% nonfat milk in Tris buffered saline Tween-20, the blots were incubated with primary antibodies: Anti-NOD2 (1:500; sc-30080; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-collagen I (1:1,000; ab34710; Abcam, Cambridge, MA, USA), anti-collagen III (1:1,000; ab7778; Abcam), anti-TGF-β (1:1,000; ab31013; Abcam) and GAPDH (1:5,000; sc-25778; Santa Cruz Biotechnology, Inc.) overnight at 4°C. Then, the membranes were incubated with horseradish peroxidase-conjugated rabbit secondary antibodies (1:2,000; K5007; Dako, Agilent Technologies, Inc., Santa Clara, CA, USA) for 1 h at room temperature. The protein bands were visualized using the SuperSignal chemiluminescent detection module (34080; Pierce, Thermo Fisher Scientific, Inc.) and images were collected using a Tanon-5200 imaging system (Tanon Science and Technology Co., Ltd., Shanghai, China).

Western blot
analysis was performed according to previous reports.^{25 (link)} The samples were prepared using the protein extraction
kit (BC3710, Solarbio) and SDS-PAGE loading buffer, separated by SDS-PAGE
gel electrophoresis, and then transferred to nitrocellulose membranes,
which were blocked with 5% nonfat milk. The membranes were incubated
with respective primary antibodies [p-Erk1/2 (1:500, #4370), Erk1/2
(1:500, #4696), cleaved caspase-3 (1:500, #9664), RIPK1 (1:500, #3493),
and β-actin (1:2000, #4970)] overnight at 4 °C. After washing
three times in TBST buffer, the membranes were incubated with respective
secondary antibodies [antirabbit IgG HRP-conjugated antibody (1:3000,
#7074) and antimouse IgG HRP-conjugated antibody (1:3000, #7076),
Cell Signaling Technology] for 2 h at room temperature. After washing
three times in TBST buffer, the membranes were visualized using a
chemiluminescent substrate (32106, Thermo Scientific) under a ChemiDoc
MP Imaging System (Bio-Rad). The optical density of the protein bands
was calculated using Image J software.

The protein sample was collected from liver tissues using a protein extraction kit (BC3710, Solarbio, China) or a Nuclear and Cytoplasmic Extraction Reagent (78833, Thermo Fisher, USA). The protein was quantified using a BCA kit (PC0020, Solarbio, China). After separating the protein by SDS-PAGE, the protein was transferred into a polyvinylidene fluoride membrane (10600023, GE Healthcare Life, USA). The membrane was blocked by 5% BSA for 1 hour, then the membrane was reacted with primary antibodies and secondary antibodies. The relative intensity of the protein bands was quantified using ana ECL kit (36208ES60, YEASEN, China) with the iBright FL1500 Imaging System (A44115, Invitrogen, USA). The results were counted by ImageJ2x (Rawak Software, Germany). The primary antibodies and secondary antibodies were as follows: anti-p53 (AF0879, Affinity, USA), anti-β-actin (AF7018, Affinity, USA), Nrf2 (AF0639, Affinity, USA), Lamin B (DF6687, Affinity, USA), Heme oxygenase-1 (HO-1, AF5393, Affinity, USA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, AF7021, Affinity, USA), and goat anti-rabbit (S0001, Affinity, USA).