Mouse monoclonal anti tgf β1

All animals were euthanized after four weeks of drug administration, and their hearts were immediately harvested and stored in liquid nitrogen until western blot analyses were performed. The following antibodies were used: mouse monoclonal anti-TGF-β1 (1: 500, ABcam, Inc.), rabbit monoclonal anti Smad3 (phosphor C25A9) (1: 500, Cell Signaling Technology, Inc.), mouse anti human Smad7 polyclonal antibody (1: 500, Cell Signaling Technology, Inc.), rabbit polyclonal anti-TNF alpha (1: 1000, ABcam, Inc.), Anti-IL6 antibody (1: 1000, ABcam, Inc.), Anti-Collagen I antibody (1: 1000, ABcam, Inc.), mouse monoclonal antialpha SMA antibody (1: 500, ABcam, Inc.), NF-κB p65 (1: 1000, Santa Cruz, Inc.) and anti-p-IκBα antibody (1: 1000, Cell Signaling Technology, Inc.). Antibody proteins were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes, which were then incubated with antibodies at 4 °C. The membranes were further incubated with horseradish peroxidase-conjugated anti-rabbit IgG (1: 2, 000) for two hours at room temperature. ECL visualization was performed and the Gene Gnome Gel Imaging System (Syngene Co.) was used to capture the resulting images. ImageJ (NIH image, Bethesda, MD) was used to analyze the gel images.