Quantitative Protein Analysis in Cardiac Fibrosis

The total protein was extracted from the cultured CFs, and the levels were compared by immunoblotting the proteins based on their expression in the left ventricular peri-infarct area (PIA) of rats [32 (link)]. Antibodies used for probing were anti-FBW7, anti-Snail, anti-SMAD3/4, anti-phosphor-SMAD3/4 (Abcam, Cambridge, UK), anti-Twist, anti-TGF-β, anti-ubiquitin, and anti-actin antibody (Sigma-Aldrich Corp. St. Louis, MO, USA). The corresponding secondary antibodies were purchased from Stressgen Biotechnologies Corporation, Victoria, BC, Canada. Signals were used for detection using the Enhanced Chemiluminescence (ECL) kit (GE Healthcare, UK). Densitometry was employed to quantify the protein levels. β-actin was used as the loading control in the western blotting experiment for normalization.