Anti p smad2

ESCs and endometrial tissues were first lysed with lysis buffer and centrifuged at 12,000 × g for 15 min at 4°C. A BCA protein assay kit (Beyotime) was used to determine the quantity of protein. Protein samples (50 μg) were separated with SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were blocked with 5% nonfat milk in TBST (10 mM Tris-HCl, 100 mM NaCl, 0.1% Tween-20, pH 7.4) for an hour at room temperature and then incubated in primary antibody overnight at 4°C. The primary antibodies used in this study were rabbit anti-β-actin (1 : 2500, control), anti-TGF-b1 (1 : 1000), anti-p-Smad2 (1 : 500), anti-Smad2 (1 : 1000), anti-p-Smad3 (1 : 1000), anti-Smad3 (1 : 500), anti-Smad4 (1 : 200), and anti-Smad7 (1 : 500) all from Santa Cruz Biotechnology. Membranes were then incubated with horseradish peroxidase-labeled goat anti-rabbit secondary antibody (1 : 10,000) for an hour at room temperature. Protein bands were visualized using Luminata Crescendo Western HRP Substrate (Millipore, Billerica, MA, USA) and a molecular imager (Bio-Rad, Philadelphia, PA, USA). Densitometry analysis was determined relative to β-actin using 1-D Analysis Software (National Institutes of Health, USA).