Ab93095

Proteins were extracted from the cardiac fibroblasts and myocardial tissues homogenized in RIPA Lysis (P0013B; Beyotime) and Extraction Buffer with a protease inhibitor cocktail, and proteins were quantified using the bicinchoninic acid method. Samples of 25-μg protein were loaded into 8% SDS-PAGE gels for electrophoresis then transferred to PVDF membranes overnight at 30V. Antibodies specific for α-SMA (dilution 1:500; ab5694; Abcam), YAP (dilution 1:100; 4912; Cell Signaling), p-YAP (dilution 1:100; 4911; Cell Signaling), and collagen I (dilution 1:1000; ab93095; Abcam) were incubated at 4°C overnight, and GAPDH (dilution 1:5000; sc66163; Santa Cruz, USA) was used as a loading control to normalize gel loading and protein expression. HRP-conjugated secondary antibodies (dilution 1:300; AS10 653; Agrisera, Sweden) plus ECL (AS16; Agrisera) were incubated at 37°C for 1 h for protein visualization. The densitometric values of bands were measured using densitometry analysis software (Multi Gauge Ver 3.0, Japan).

Protein lysates were prepared from tissue cryosections (10 times, 10 μm) in RIPA buffer (Thermo Scientific) supplemented with 1% protease inhibitor cocktail (Sigma-Aldrich). Lysates were homogenized using sonication. Protein concentrations were determined using a DC protein assay (Bio-Rad, Hercules, CA, USA) after which 8μg of protein was spotted in triplicate on a Tris buffered saline (TBS) pre-soaked nitrocellulose membrane using a 96-well dot-blot apparatus (Bio-Rad). To reduce non-specific binding, spotted membranes were blocked in TBS + 0.5% Tween20 (Sigma-Aldrich) containing 5% milk powder. Spotted membranes were stained with primary antibodies directed against Collagen type I (1:1000, ab93095, Abcam) or Collagen type III (1:2000, ab6310, Abcam) diluted in TBS-Tween20 containing 5% milk powder. All washing steps were performed in TBS-Tween20. Protein bands were visualized using chemiluminescence and a ChemiDoc imaging system (Bio-Rad). Image analysis was performed using the Dot Blot analyser plugin for ImageJ, whereby collagen expression of control rats at time of biopsy was used as a reference.