Western Blot Analysis

The procedures for sample preparation and Western blot were conducted as described in our previous studies (Liu et al., 2014; Li et al., 2019a; Wang et al., 2019). Briefly, the RV tissues, H9c2 cells, or cardiac fibroblasts were homogenized with ice-cool lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, and 1% Triton-X-100) with a protease and phosphatase inhibitor cocktail (Beyotime, Shanghai, China). And then, the protein concentration was detected according to the BCA assay kit instructions (Beyotime, Shanghai, China). Samples containing 30–40 μg of protein were subjected to 8 or 10% SDS-PAGE gel, and then they were transferred to polyvinylidene fluoride (PVDF) blotting membranes (G.E. Healthcare, Germany). The PVDF membranes were incubated with primary antibodies against collagen Ⅰ (Abcam, Cambridge, MA, USA), α-SMA (Cell Signaling Technology, Massachusetts, USA), p-JAK2 (Abcam, Cambridge, MA, USA), JAK2 (Beyotime, Shanghai, China), p-STAT3 (Signalway Antibody, Maryland, USA), STAT3 (Signalway Antibody, Maryland, USA), and α-tubulin (Santa Cruz, Texas, USA) followed by horseradish peroxidase (HRP) conjugated secondary antibody (Beyotime, Shanghai, China). The signals of Western blot bands were detected by BeyoECL Moon kit (Beyotime, Shanghai, China) through Molecular Imager ChemiDoc XRS System (Bio-Rad, Philadelphia, USA). Densitometric quantification was carried out by Image J (NIH, USA). The α-tubulin served as a loading control.