3 mst

The expressions of hepatic CSE and 3-MST proteins and β-actin (house-keeping protein) were determined by western blot technique. To be brief, hepatic expression of these proteins on the nitrocellulose membranes were detected using CSE (1:1000; Abnova, USA), 3-MST (1:1000; Santa Cruz, The Netherlands) and β-actin (1:5000; Abcam, Canada) primary antibodies following an overnight incubation at 4 °C. Next, the membranes were washed three times with a washing buffer (Tris-buffered saline [TBS] with 0.004% Tween) and incubated with their respective HRP-linked secondary antibodies (1:1000) in TBS + Tween-20 supplemented with 3% bovine serum albumin (w/v) at room temperature for 1 h. The blots were developed with SuperSignal West Dura Extended Duration Substrate (Thermo Scientific, USA) and protein bands were visualized with a Gene Genome. The intensities of the target protein bands were quantified using GeneTools analysis software (Westburg B.V., Leusden, The Netherlands) and normalized against the intensity of β-actin.

The kidney cytoplasm nuclear proteins were collected under the kit protocol (Nuclear and Cytoplasmic Protein Extraction Kit, Beyotime Biotechnology, Nanjing) and quantified using a BCA reagent (Shen Neng Bo Cai Corp, Shanghai). The proteins were resolved on a sodium dodecyl sulfate 10% polyacrylamide gel and transferred onto polyvinylidene fluoride membrane (Millipore, Bedford, MA, USA) and incubated with primary antibodies (1 : 1000 dilution) against Bcl-2, Bax, CSE, CBS, 3-MST, SIRT1 (Santa Cruz, CA, USA), Collagen I (Col I), Collagen III (Col III), Fibronectin (FN), SOD1, SOD2 (Abcam Company, USA), or Nrf2, HO-1 (Proteintech, China) at 4°C overnight. The blots were washed with phosphate buffer saline (TBST) for three times and then incubated with horseradish peroxidase-conjugated secondary antibodies for another 1 hour at room temperature. After washing, the blots were visualized by using chemiluminescent substrate (ECL). The densities of immunoblot bands were analysed using a scanning densitometer (model GS-800, Bio-Rad Laboratories, Hercules, CA, USA) coupled with Bio-Rad personal computer analysis software.

Frozen heart tissues were lysed mechanically in cold RIPA buffer. Proteins were extracted and quantified using a BCA protein assay kit (Best Bio, Shanghai, China). The proteins were electrophoretically separated using SDS-PAGE and transferred to a polyvinylidene difluoride membranes (Millipore, United States). The membranes were blocked with 3% BSA for 1.5 h at room temperature, and antigens were detected using the following antibodies at 4°C overnight, CSE (1:1,000, Proteintech, United States), CBS (1:1,000, Proteintech, United States), 3-MST (1:5,000,SantaCruz, United States), P53 (1:2000, Proteintech, United States), P21 (1:2000, Proteintech, United States), NRF2 (1:2000, Proteintech, United States), SLC7A11 (1:2000, Proteintech, United States), ACSL4 (1:2000, Abcam, United States), GPX4 (1:2000, Proteintech, United States), FPN1 (1:1,000, Proteintech, United States), FTH (1:1,000, Immunoway, United States), TRF1 (1:1,000, Abcam, United States), and GAPDH (1:5,000, Proteintech, United States). The blots were incubated with a horseradish peroxidase-conjugated anti-rabbit (Proteintech, United States) or anti-mouse (Proteintech, United States) secondary antibody at room temperature for 1.5 h. All antibodies were diluted with TBST. Blot bands were visualized by enhanced chemiluminescence and detected by ImageJ software (1.52, NIH, United States).