Anti smad2

Protein samples were separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA). After blocking at room temperature in 5% w/v nonfat milk with TBST buffer (Tris-HCl 10 mM, NaCl 120 mM, and Tween-20 0.1%; pH 7.4) for 2 h, membranes were incubated overnight with the appropriate primary anti-collagen I, anti-collagen III, anti-TGF β1 (1 : 1000, Santa Cruz Biotechnology, Santa Cruz, CA), anti-Smad2, anti-p-Smad2 (1 : 500, Bioworld Technology, St. Louis Park, MN), or anti-GAPDH (1 : 6000, Sigma-Aldrich, St. Louis, MO) antibodies, at 4°C, followed by horseradish peroxidase- (HRP-) conjugated secondary antibody at room temperature for 2 h. Proteins were visualized by enhanced chemiluminescence substrate (ECL, Pierce, Rockford, IL).

Protein samples were separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA). After blocking at room temperature in 5% w/v non-fat milk with TBST buffer (Tris-HCl 10 mM, NaCl 120 mM, Tween-20 0.1%; pH 7.4) for 2 h, membranes were incubated overnight with the appropriate primary anti-collagen I, anti-collagen III, anti-TGF β, anti-Smad2, anti-p-Smad2, anti-CBP, anti-Ser142-p-CREB, anti-Ser133-p-CREB (1∶500, Bioworld Technology, St. Louis Park, MN), anti-tubulin, anti-Lamin B1(1∶1000, Santa Cruz Biotechnology, Santa Cruz, CA), anti-GAPDH (1∶6000, Sigma-Aldrich, St. Louis, MO) at 4°C and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 2 h. Proteins were visualized by enhanced chemiluminescence substrate (ECL, Pierce, Rockford, IL).

Protein preparation and western blot were performed as described previously [19 (link)]. The antibodies for immune blot analysis were as follows: mouse anti-Flag M2 (Sigma-Aldrich), rabbit anti-DACH1 (Proteintech), rabbit cyclinB1 (Bioworld Technology) and rabbit cdc2 (Bioworld Technology), rabbit anti-phospho-SMAD3 (Cell Signaling Technology, Inc, Shanghai, China), rabbit anti-phospho-SMAD2 (Millipore, Billerica, MA, USA), rabbit polyclonal anti-SMAD3, anti-SMAD2, anti-E-cadherin, anti-vimentin, anti-MMP-2, anti-MMP-9 (Bioworld Technology). The bands were visualized by enhanced chemiluminescence (Pierce Bioscience, Shanghai, China).