P smad2 3 antibody

Cells or liver homogenates were harvested and washed twice in cold PBS and then were treated with RIPA lysis buffer (Beyotime, Shanghai, China) on ice for 30 min. The lysate was collected into micro-tubes and centrifuged for 15 min at 12,000 rpm at 4 °C. Protein samples (20 mg) were denatured with 5× SDS loading buffer at 100 °C for 5 min, and then were segregated on 10% SDS polyacrylamide gel electrophoresis and transferred onto 0.45-mm nitrocellulose membranes. The membranes were cut into bands of appropriate width according to the protein marker and after 60 min of blocking with 5% fat-free milk, membranes were incubated with Smad 7 antibody (1:2000; Sigma, USA), COLI antibody (1:2000; Sigma, USA), P-Smad2/3 antibody (1:1000; Abclonal, Wuhan, China), COLIII antibody (1:1000, Abclonal, Wuhan, China), α-SMA (1:1000, Abclonal, Wuhan, China), and β-actin antibody (1:2000; Abmart, Shanghai, China) overnight at 4℃, correspondingly. Blots were washed for 1 h with the anti-rabbit secondary antibody (1:2000; Cell Signaling Technology, USA). After washing three times with TBST, immunoreactive protein bands were detected using enhanced chemiluminescence reagents (Bio-Rad, CA, USA). Band intensities were normalized to β-actin and analyzed using ImageJ software.