Digital gel imaging system

All proteins were separated by SDS-PAGE and then electroblotted onto PVDF membranes (Millipore, USA) before blocking with 5% (w/v) skimmed milk (BD, USA) for 1 h. The blocked PVDF membrane was incubated with diluted primary antibody in blocking buffer at 4° C overnight. The next day, the PVDF membrane was rinsed and incubated at room temperature with diluted HRP-conjugated secondary antibodies such as anti-mouse-HRP (Abcam, USA) or anti-rabbit-HRP (Abcam, USA) for 2 h. ECL luminescent solution (Meilunbio, China) was used to visualize bound antibodies using a digital gel imaging system (Biorad, USA). The antibodies used are: mouse anti-β-Actin used at 1:5000 (CST, USA; #4970S), FSTL3 used at 1:1000 (Abcam, USA; #ab232761).

For direct detection of protein levels, the heart was quickly cut off after 120 min of reperfusion; total protein was obtained from the heart tissue according to the manufacturer's instructions. Protein concentration was determined by BCA Protein Assay Kit (KeyGen BioTECH, China). 30 ug of total proteins was separated by electrophoresis on SDS-PAGE and then transferred onto 0.45 μm PVDF membranes. The membranes were blocked for 2 h in 5% skim milk at room temperature and incubated with primary antibodies at 4°C overnight. Immunoblots were performed using the following antibodies: RAGE (Cell Signaling Technology, USA), HMGB1 (Cell Signaling Technology, USA), phosphorylated ERK1/2 (Cell Signaling Technology, USA), total ERK (Jiancheng Technology, CHINA), phosphorylated Akt (Cell Signaling Technology, USA), total Akt (Jiancheng Technology, CHINA), and rabbit anti-GAPDH (Bioworld, China). Then the membranes were washed with TBST. We blocked the protein with phosphate-horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology, USA) at room temperature for 2 hours. The complexes were detected by using ECL kit (Thermo, USA). The images were analyzed with digital gel imaging system (Bio-Rad, USA).