Ab134966

Total protein was extracted from H9C2 cells, heart, liver, spleen, lungs, and kidney tissues using the RIPA lysis buffer (Beyotime, Beijing, China) containing protease and phosphatase inhibitors. Protein concentrations were measured using the BCA Protein Assay Kit (Beyotime). Protein was separated by SDS-PAGE (10 % gels) and transferred onto 0.22 μm polyvinylidene fluoride (PVDF) membranes. Each membrane was then incubated at 4°C overnight with primary antibodies against SAHH (1:5,000; ab134966; Abcam, Shanghai, China), p-AMPK (1:5,000; ab133448; Abcam, Shanghai, China), AMPK (1:5,000; ab32047; Abcam, Shanghai, China), FOXO3 (1 μg/ml; ab23683; Abcam, Shanghai, China), SIRT3 (1:1,000; ab189860; Abcam, Shanghai, China), BAX (1:1,000; ab182734; Abcam, Shanghai, China), Bcl-2 (1:2,000; ab194583; Abcam, Shanghai, China), cleaved caspase 9 (1:1,000; AF5240; Affinity, Jiangsu, China), and cleaved caspase 3 (1:1,000; AF7022; Affinity, Jiangsu, China). After incubation with HRP-labeled secondary antibodies (1:10,000; ab6721; Abcam, Shanghai, China) at 37°C for 1 h, protein bands were visualized using a chemiluminescence imaging system (Tanon, Shanghai, China).

Total protein was extracted from cells using radio-immunoprecipitation assay (RIPA) buffer (Sigma-Aldrich). 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis was used for protein separation. The separated proteins were transferred to polyvinylidene flouride membranes (Millipore). Then, the membranes were blocked with 5% skim milk for 1.5 h. After incubation with the primary antibodies at 4°C overnight, membranes were incubated with the secondary antibodies at room temperature for 1 h. Visualization of proteins was carried out using ImageJ software (Bio-Rad, Hercules, CA). Anti-AHCY (1:1000, ab134966, Abcam, MA, USA), anti-KCMF1 (1:1000, ab192761, Abcam), anti-MANBAL (1:1000, ab224611, Abcam) and anti-β-actin (1:1000, ab181092, Abcam) were implemented as the primary antibodies. Goat Anti-Rabbit IgG H&L (1:5000, ab96899, Abcam) and goat Anti-Mouse IgG H&L (1:5000, ab96879, Abcam) were used as the secondary antibodies.