Bio id software

Total protein was extracted from heart tissues using RIPA lysis buffer (150 mmol/L NaCl, 1% Triton X‐100, 1% sodium deoxycholate, 50 mmol/L Tris‐HCl at pH 7.5, 2 mmol/L EDTA, 1 mmol/L PMSF, 1 mmol/L DTT, 1 mmol/L Na₃VO₄ and 5 mmol/L NaF) containing a protease inhibitor cocktail (Calbiochem/EMD Millipore, Billerica, MA, USA). Proteins were subjected to SDS‐PAGE and transferred to polyvinylidene difluoride membranes, which were then blocked with 5% skim milk in TBST buffer (20 mmol/L Tris, 200 mmol/L NaCl and 0.04% Tween‐20) for 1 h at 25°C. The membranes were incubated overnight at 4°C with primary antibodies against collagen type III, fibronectin, connective tissue growth factor (CTGF), atrial natriuretic peptide (ANP), GATA4, Sp1, and GAPDH, before being exposed to anti‐rabbit or anti‐mouse horseradish peroxidase‐conjugated secondary antibodies (diluted 1:5000) for 1 h at 25°C. Protein bands were visualized using Immobilon western blotting detection reagents (EMD Millipore). Bio‐ID software (Vilber Lourmat, Eberhardzell, Germany) was used to quantify protein expression.

At specific time-points after IC or after I/R, tissues were collected and homogenized in lysis buffer containing 25 mM Hepes (pH 7.5), 1% Triton X100, 150 mM NaCl, 10% glycerol, 1 mM sodium orthovanadate, 50 mM NaF, 10 mM sodium pyrophosphate, and protease inhibitor cocktail (Sigma Aldrich, St Louis, MO). Equal amounts of proteins were separated by denaturing SDS-PAGE, and then transferred onto nitrocellulose membrane (Thermo Fisher Scientific, Waltham, MA). These membranes were probed using primary antibody, and then incubated with HRP-conjugated secondary antibody. Protein signals were detected using the ECL plus system (GE Healthcare, Chicago, IL). Expression levels were determined from band intensities using Bio-ID software (Vilber Lourmat, Collégien, France), and values were expressed relative to GAPDH signals [30 (link), 31 (link), 37 (link)]. From Cell Signaling Technology (Beverly, MA), we purchased antibodies against phosphorylated STAT3 (P-STAT3; #9145, D3A7, rabbit monoclonal, 1:200 dilution), phosphorylated AKT (P-AKT; #4060, D9E, rabbit monoclonal, 1:200 dilution), and phosphorylated ERK1/2 (P-ERK1/2; #4370, D13.14.4E, rabbit monoclonal, 1:200 dilution). The antibody against glyceraldehyde 3-phosphate dehydrogenase (GAPDH; MAB374, 6C5, mouse monoclonal, 1:5000 dilution) was purchased from Merck Millipore (Darmstadt, Germany).

Total protein was extracted from heart tissues using RIPA lysis buffer (150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 50 mM Tris-HCl at pH 7.5, 2 mM EDTA, 1 mM PMSF, 1 mM DTT, 1 mM Na₃VO₄, and 5 mM NaF) containing a protease inhibitor cocktail (Calbiochem/EMD Millipore, Billerica, MA, United States). Proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride membranes, and blocked with 5% skim milk in TBST buffer (20 mM Tris, 200 mM NaCl, and 0.04% Tween 20) for 1 h at 25°C. Next, the blots were incubated with primary antibodies overnight at 4°C, followed by anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibodies (diluted 1:5,000) for 1 h at 25°C. Protein bands were visualized using Immobilon western blotting detection reagents (EMD Millipore, Billerica, MA, United States). Bio-ID software (Vilber Lourmat, Eberhardzell, Germany) was used to quantify protein expression.