Erk1 2

Tissues from the LV infarct border area (5 mm) and gastrocnemius were homogenized. Total proteins extraction and SDS-PAGE were performed as described before58 (link). The dilution of primary antibodies as follows: FSTL1 (1:1000, GeneTex, Irvine, CA, USA), TGFβ1 (1:500, Bioworld Technology, St. Louis, MN, USA), Smad2/3 (1:1000, Bioworld), p-Smad2/3 (1:500, Bioworld), Akt (1:2000, Cell Signaling, Danvers, MA, USA), p-Akt (1:2000, Cell Signaling), Erk1/2 (1:500, Signalway Antibody, College Park, MD, USA), p-Erk1/2 (1:500, Signalway Antibody), AMPKα1 (1:500, Signalway Antibody), p-AMPKα1 (1:500, Signalway Antibody), Smad1/5/8(1:500, Signalway Antibody), p-Smad1/5/8(1:1000, Cell Signaling) and GAPDH (1:10000, Bioworld). Following incubating with horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000 dilution, Jackson ImmunoResearch, West Grove, PA, USA), protein bands were subsequently developed with enhanced chemiluminescence. Western quantification was performed by Image Processing and Analysis in Java 1.48 (Wayne Rasband, National Institutes of Health, USA).

Cells were lysed with RIPA lysis buffer and Phenylmethyl sulfonyl fluoride (PMSF, A610425, Sangon Biotech). The protein concentration was determined by a BCA protein assay kit (Thermo). Equal amount of protein samples in SDS sample buffer (1% SDS, 62.5 mM Tris-HCl, pH 6.8, 10% glycerol, 5% mercaptoethanol, and 0.05% bromphenol blue) were boiled for 5min and subjected to reducing 10% SDS-PAGE. Proteins were separated by SDS-PAGE and electrophoretically transferred to PVDF membrane. The membrane was blocked with 5% non-fat milk powder in PBST at room temperature for 2h, and then incubated with primary antibodies at 4℃ overnight. Following washing, the membrane was incubated with secondary antibody conjugated with horseradish peroxidase (anti-rabbit or anti-mouse) at room temperature. The following antibodies were used: antibodies against phosphor-MAPKAPK-2 (Thr334) (#3041, 1:1000), and AKT (9272S, 1:1000) were purchased from Cell Signaling Technology; antibodies against AKT (phospho-ser473) (11054, 1:1000), ERK1/2 (phospho-Thr202) (12082, 1:1000) and ERK1/2 (29162, 1:1000) were purchased from Signalway Antibody; antibody against V5-tag (66007-1-Ig, 1:1000) was purchased from Proteintech.

Blots in PVDF membranes were incubated overnight with primary antibody at 4°C. AKT (1:1000), p-AKT (1:1000), ERK1/2 (1:1000), p-ERK 1/2 (1:1000), SAPK/JNK(1:1000), p-SAPK/JNK(1:1000), p-P38MAPK(1:1000) (Signalway Antibody LLC, Maryland, USA), CXCR3 (1:600, ABGENT Company, San Diego, USA), CXCR3-A and CXCR3-B (1:500, BOSTER Company, Wuhan, China), followed by anti-rabbit IgG (1:3,000; Sigma, CA) for 1 h at room temperature, and the signals were detected with an ECL assay kit (Amersham, Buckinghamshire, UK).