Gal 3

Total proteins from human cardiac fibroblast were separated by SDS-PAGED on 10% polyacrylamide gels and transferred to Hybond-c Extra nitrocellulose membranes (Amersham Biosciences, Piscataway, NJ). Membranes were probed with primary antibodies for RCN-1 (Abcam; dilution 1:500), RCN-2 (Abcam; dilution 1:500), RCN-3 (Abcam; dilution 1:500), collagen I (Sigma; dilution 1:500), collagen III (Santa Cruz; dilution 1:500), Fibronectin (Millipore; dilution 1:1000), Gal-3 (ThermoFisher; dilution 1:1000), CT-1 (Abcam; dilution 1:500), connective tissue growth factor (CTGF; Torrey Pines Biolabs Inc., dilution 1/1000), ERK1/2 and ERK1/2-P (Thr202/Tyr204) at 1/1000 (Cell Signaling), Akt and Akt-P (Ser473) at 1/1500 (Cell Signaling), Stat3 and Stat3-P (Tyr705) at 1/1500 (Cell Signaling). Western blots were performed with stain-free gels for loading control. After washing, detection was made through incubation with peroxidase-conjugated secondary antibody, and developed using an ECL chemiluminescence kit (Amersham). After densitometric analyses, optical density values were expressed as arbitrary units. Results are expressed as an n-fold increase over the values of the control group in densitometric arbitrary units. All Western Blots were performed at least in triplicate for each experimental condition.

Aliquots of 20 µg of total proteins were prepared from cells and mitral valves and electrophoresed on SDS polyacrylamide gels and transferred to Hybond-c Extra nitrocellulose membranes (Bio-Rad). Membranes were incubated with primary antibodies for Vimentin (1:100, Santa Cruz Biotechnology), α-Smooth Muscle Actin (α-SMA, 1:100, Sigma-Aldrich), Fibronectin (1:100, Merck Millipore, Darmstadt, Germany), Elastin (1:50, Abcam), Gal-3 (1:100, Thermo Fisher Scientific, Waltham, Massachusetts, USA), CD31 (1:100, Abcam), vWF (1:100, Santa Cruz Biotechnology), VE-cadherin (1:100, Abcam). Stain free and β-Actin (Sigma-Aldrich) detection was used as loading control. After washing, detection was made through incubation with peroxidase-conjugated secondary antibody, and developed using an ECL chemiluminescence kit (Amersham, GE healthcare, Thermo Fisher Scientific, UK). After densitometric analyses, optical density values were expressed as arbitrary units. All Western blots were performed at least in triplicate for each experimental condition. Whole original images are displayed in the figures.