Gtx111010

The frozen heart tissue samples were thawed. Protein concentrations were determined using a BCA protein assay kit (Multisciences, Shanghai, China). Samples were mixed with 1× loading buffer and denatured at 95°C for 10 min. We resolved 30 ug of protein by sodium dodecyl sulfate polyacrylamide gel electrophoresis on a 10% or 15% SDS-PAGE and electrophoretically transferred it to a polyvinylidene difluoride (PVDF) membrane. After blocking with 5% milk in TBST for 4 h at room temperature, the rat monoclonal antibodies specific for rat Opa1 (A9833, ABclonal, USA), Mfn1 (A9880, ABclonal, USA), Mfn2 (A5750, ABclonal, USA), Drp1 (A2586, ABclonal, USA) and Fis1 (GTX111010, GeneTex, USA) and alpha Tubulin (GTX628802, GeneTex, USA) were incubated at a concentration of 1: 500 (for Opa1, Mfn1, Drp1), or 1: 1000 (for Mfn2, Fis1, and Tubulin) in 1×TBST overnight at 4°C. The infrared-labeled anti-rat secondary antibodies (26055, Rockland, USA) at a concentration of 1: 10 000 were added to PVDF membranes and incubated for 2 h at 37°C. The membranes were scanned and the band densities were quantified using the Odyssey Infrared Imaging System.

The total proteins and mitochondrial proteins were extracted from heart tissues using the total protein extraction kit (KGP2100) and mitochondrial protein extraction kit (KGP8100) from KeyGEN BioTECH. The extraction processes were conducted in an ice-water bath according to the instructions of the kits. The protein concentration was determined using the BCA Protein Assay Kit (KeyGEN BioTECH, KGP8100). Equal amounts of protein (20 µg) from each animal subjected to heat denaturation were loaded for electrophoresis and transferred onto nitrocellulose membranes. The membrane was blocked with 10% skim milk at room temperature and incubated overnight at 4°C with the following primary antibodies: Drp1 (1:1000, ab184247, Abcam), phospho-Drp1 (Ser616 1:1000, 3455, Cell Signaling), Fis1 (1:1000, GTX111010, GeneTex), LC3B (1:2000, ab192890, Abcam) and VDAC (1:1000, GTX114187, GeneTex). The membranes were washed and incubated with horseradish peroxidase-labeled secondary antibodies for 1 hour at room temperature, followed by imaging using the Pierce ECL Western Blotting Substrate. The VDAC bands served as a mitochondrial and GAPDH as cytosolic internal controls. The ratios of targeted protein bands to loading controls were measured and presented as relative target band intensity.