Mfn1 2

Proteins collected from myocardial tissues and H9C2 cells were lysed by RIPA lysate (Beijing Applygen Technolog Inc., China) according to the manufacturer's instruction. The protein content was quantified using the BCA method. After addition of loading buffer and boiling for 5 minutes at 99°C, protein samples (25 mg per sample) were separated by 10% Tris/Glycine SDS-PAGE and then electrotransferred to PVDF membranes at 300 mA for 1.5 h. Membranes were incubated in TBST containing 5% nonfat milk for 1 hours. Then the membranes were incubated with different primary antibodies including rabbit monoclonal antibodies against MFN1/2 (Proteintech), OPA1 (Proteintech), FIS1 (Proteintech), DRP1 (Proteintech), PGC-1α (Proteintech), NRF1(Proteintech), TFAM (Proteintech), and GAPDH (Proteintech) overnight at 4°C. The blots were washed with TBST three times and then incubated with anti-rabbit IgG (1 : 2000) for 4 h at 4°C. After washing three times with TBST, the immunolabeling was detected using Omni ECL reagent (EpiZyme, China) for 1 min at room temperature. The ultimate expression of every single protein was standardized by GAPDH. The band grayscale analysis was performed using Image J software.

After euthanasia, the liver and pancreas were processed, including 10% neutral formaldehyde fixing, paraffin embedding, 5 µm thickness sectioning, and H&E staining [26 (link)]. The pathological changes were observed using a stereomicroscope (SMZ25, Nikon, Tokyo, Japan). For immunohistochemistry, the sections of the liver were antigen retrieval, blocked with goat serum (37 °C for 20 min), and then incubated with primary antibodies. The antibodies included AMPK (1:100, Beyotime, Shanghai, China), Phosphorylated protein kinase RNA-like endoplasmic reticulum kinase (p-PERK, 1:200, Bioss, Beijing, China), Dynamic-related protein 1 (DRP1, 1:100, Beyotime), Cytochrome c (CytC, 1:100, Proteintech, Chicago, IL, USA), caspase 12 (1:100, Bioss), Inositol-requiring enzyme-1 (IRE1, 1:100, Proteintech) and Mitofusion 1/2 (MFN1/2, 1:100, Proteintech). Subsequently, the slides were visualized using DAB and then observed under a stereomicroscope (SMZ25, Nikon).