Rabbit monoclonal to smad3

Cells were incubated on the coverglasses in six orifice plates, and then fixed with 4% paraformaldehyde. Antibodies and dilutions were as follows: rabbit monoclonal to smad3 (1:500; Abcam) and rabbit monoclonal to smad7 antibody (1:500, Epitomics). The cells were then incubated with the secondary antibody for two hours. DAPI was used to stain the cell nuclei (blue). Cells were observed under the confocal microscope (Leica TCS SP5 MP, Heidelberg GmbH,German). For in vivo studies, kidney tissue sections (4μm) were subjected to immunohistochemical staining (IHC) for smad3 and smad7. The percentage of positively-stained area with 40 fields of view was analyzed by Image-pro plus 6.0 (Media cybernetics).

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)].

Protein samples were subjected to 10% SDS-polyacrylamide gel electrophoresis and then transferred to PVDF membranes. The PVDF membranes were blocked with 50% skimmed milk, treated with primary antibody at 4°C overnight, washed and then incubated with the secondary horseradish for two hours. Bands were detected with Enhanced Chemiluminescence (ECL). Immunoblotting was performed with rabbit polyclonal to TGF-β1 antibody (Abcam, 1:200), rabbit monoclonal to smad3 (1:500; Abcam), rabbit monoclonal to rabbit monoclonal to smad7 antibody (1:500, Epitomics). Then, membranes were incubated with the secondary horseradish (1:5000) and exposed to X-ray. Densitometry was detected by Imagine J. Western blot analyses were performed at least in triplicate.