Sirt1

RIPA buffer containing 100 μM PMSF (Solarbio) is used to lyse cardiomyocytes and cardiac tissues. Cardiomyocytes or cardiac tissues were suspended in RIPA buffer and were lysed on ice for 20 min. The lysate was centrifuged at 4 °C, 13,000 rpm for 20 min, and the concentration of total protein was measured by BCA kit (Vazyme). SDS-PAGE electrophoresis was used to separate cell lysates, which were then transferred onto PVDF membranes. The primary antibody was incubated with the membrane at 4 °C overnight, the blot was incubated with HRP-conjugated secondary antibodies for 1 h at room temperature followed by development with ECL Western blot substrate. We use primary antibodies in the following concentrations: GPX4 (ZEN-BIOSCIENCE, 381958) 1:3000, SLC7A11 (ZEN-BIOSCIENCE, 382036) 1:3000, FTH1 (ZEN-BIOSCIENCE, 381204) 1:2000, NAMPT (Klean AB, P100462) 1:3000, Sirt1 (Zenbio, R25721) 1:2000, FOXO1 (Zenbio, 380978) 1:1000, Ac-FOXO (Affinity, AF2305) 1:1000, RIPK1(Affinity, AF7877) 1:2000, RIPK3 (Affinity, DF10141) 1:2000, MLKL (Affinity, DF7412) 1:2000, BAX (Klean AB, P100045) 1:1000 and GAPDH (Abclonal, A19056) 1:5000, overnight at 4 °C.

The specific process followed the previous article.
³⁵ (link) Briefly, proteins were isolated from cells by using RIPA lysis buffer and separated by 10% SDS‐polyacrylamide gel electrophoresis. Then they are transferred to PVDF membranes. Thereafter, the membranes were incubated with primary antibodies against β‐Actin (sc‐47778, Santa Cruz Biotechnology), Mfn1 (No. 509880, ZEN BIO), Mfn2 (No. 340604, ZEN BIO), Opa1 (No. 382025, ZEN BIO), phospho‐AMPKα1 (No. R26252, ZEN BIO), AMPKα1 (No. 380431, ZEN BIO), PGC1α (No. 381615, ZEN BIO), SIRT1 (No. R25721, ZEN BIO), Smad3 (#ab28379, Abcam), phospho‐Smad3 (No. R22919, ZEN BIO), Smad4 (#ab40759, Abcam), Drp1 (No. 221099, ZEN BIO) and Fis1 (No. R26001, ZEN BIO) at 4°C overnight, and the secondary antibodies against rabbit (sc‐2357, Santa Cruz Biotechnology) and mouse (sc‐516102, Santa Cruz Biotechnology) were incubated for 1 h at room temperature. Immobilon® Western kit (P90719, Millipore, Massachusetts, USA) was used to visualize the protein bands.