Immobilon western blotting detection reagents

Total proteins were extracted from the cardiac tissues or cardiac fibroblasts using radioimmunoprecipitation assay lysis buffer (150 mM NaCl, 1% Triton X‐100, 1% sodium deoxycholate, 50 mM Tris–HCl [pH 7.5], 2 mM ethylenediaminetetraacetic acid, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 1 mM Na₃VO₄ and 5 mM NaF) supplemented with a protease inhibitor cocktail (Calbiochem/EMD Millipore). The protein concentration was determined using the Pierce bicinchoninic acid protein assay kit (Thermo Fisher Scientific). Equal amounts (40 μg) of proteins were subjected to sodium dodecyl sulfate‐polyacrylamide gel electrophoresis using a 10% gel. The resolved proteins were transferred onto a polyvinylidene difluoride membrane (pore size = 0.45 μm), and the membrane was blocked with 5% skim milk and incubated with the primary antibodies (1:1000) at 4°C overnight. Next, the membrane was washed three times with Tris‐buffered saline containing Tween 20 buffer (20 mM Tris, 200 mM NaCl and 0.04% Tween 20) to remove unbound proteins. It was then incubated with anti‐rabbit or anti‐mouse horseradish peroxidase–conjugated secondary antibodies (1:5000) for 1 h at 25°C. Immunoreactive signals were visualized using Immobilon Western blotting detection reagents (EMD Millipore), and the protein band intensities were quantified using ImageJ (National Institutes of Health).

Total proteins were isolated from the cardiac and pulmonary tissues using a radioimmunoprecipitation assay buffer as described previously [28 (link)]. The protein concentration was measured using the bicinchoninic protein assay kit. Equal amounts of proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The resolved proteins were transferred to a polyvinylidene difluoride membrane (pore size: 0.45 μm; Merk Millipore, MA, USA). The membrane was blocked with 5% skim milk in Tris-buffered saline containing Tween-20 (TBST) (20 mM Tris, 200 mM NaCl, and 0.04% Tween 20) for 1 h at 25°C and probed with the primary antibodies (1 : 1000) overnight at 4°C. Next, the membrane was washed thrice with TBST for 5 min and incubated with the anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibodies (1 : 3000) for 1 h at 25°C. Immunoreactive signals were detected using Immobilon western blotting detection reagents (EMD Millipore, Billerica, MA, USA). The intensities of the protein bands were quantified using ImageJ software (https://imagej.net/).

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.