Western Blot Analysis of TGF-β Signaling

TGF-β1, E-CA, α-SMA, smad3/p-smad3, and smad7 were detected by western blot as previously described [24 (link), 25 (link)]. Western blot was performed with mouse monoclonal to TGF-β1 (Abcam, 1 : 500), rabbit monoclonal to E-CA (CST, 1 : 500), rabbit polyclonal to α-SMA (Abcam, 1 : 500), rabbit monoclonal to smad3 (Abcam, 1 : 500), rabbit polyclonal to p-smad3 (CST, 1 : 500), and rabbit polyclonal to smad7 (Epitomic, 1 : 500). The membranes were then incubated with horseradish peroxidase-conjugated secondary antibody (1 : 5000). Western blot analyses were performed at least in triplicate. Densitometry was detected by Image J [26 (link), 27 (link)].