Chi3l1

50 mg of heart tissues removed from the border zones of infarct area were cut and homogenized in RIPA buffer (Cat# P0013B, Beyotime Biotechnology, Shanghai, China) added with protease inhibitor cocktail (Cat# 78430, Thermo Scientific, Waltham, MA, USA), and centrifuged at 12,000 rpm for 30 min at 4 °C. The supernatant was collected and the protein concentration was quantified using a BCA protein assay (Cat# P0012, Beyotime Biotechnology, Shanghai, China). For western blotting experiments, protein samples were first separated with SurePAGE gels (Cat# M00656, GenScript, Nanjing, China) and transferred to a nitrocellulose membrane (Millipore, Burlington, MA, USA), which was incubated with one of the following primary antibodies in the indicated concentrations: CHI3L1 (1:500, #ab77528, Abcam, Cambridge, MA, USA), PAR2 (1:500; #YN2681, Immunoway Biotechnology, Plano, TX, USA), PI3K (1:1000, #4249, Cell Signaling Technology, Danvers, MA, USA), p-PI3K (1:500; #17366, Cell Signaling Technology, Danvers, MA, USA), ERK (1:1000, #4695, Cell Signaling Technology, Danvers, MA, USA), pERK (1:500, #4370, Cell Signaling Technology, Danvers, MA, USA), eNOS (1:800; #ab16798, Abcam, Cambridge, MA, USA), and GAPDH (1:3000, #TDY042, TDY biotech, Beijing, China).

After OSM and STAT3 inhibitor treatment, the total proteins were extracted by RIPA buffer (Pierce, Rockford, IL) on ice. Equal quantities of protein samples were separated by electrophoresis on 12% SDS-PAGE polyacrylamide gels. Then, the samples were electro-transferred to PVDF membranes (0.22 μm, Millipore) using a wet transfer apparatus (Bio‐Rad) and blocked with 5% BSA in PBS for 1 h at room temperature. The membranes were incubated overnight at 4 °C with primary antibodies of CD44, FN1, CHI3L1, CD24, DLL3, OLIG2 and β-ACTIN (Abcam), respectively. After that, the blots were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (#HA1001, huabio, 1:5000) at room temperature for 1 h [30 (link)]. The immobilon reagents (Millipore) was used for the visualization and detection of antibody-antigen complexes. The band intensity was measured by ImageJ software.