Gd cz5616 r

Total cellar protein extraction from different treated MEPM cells using 5 × sodium dodecyl sulfate-lysis buffer supplemented with protease inhibitors (M250; Amresco, Ohio). Protein concentration was determined using a standard BSA protein assay (DingGuo, Beijing, China). Then 40 μg of proteins were fractionated on 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. After blocking with 5% nonfat milk, the membranes were immunoblotted with the primary antibodies: Smad2 (ab71109; Abcam, Massachusetts), p-Smad2 (sc101801; Santa Cruz, California), Smad3 (BM3559; Boster Biotech, Wuhan, China), p-Smad3 (GD-CZ5616 R, Santa Cruz, California), Smad4 (PB0446; Boster Biotech, Wuhan, China), and Smad7 (sc-365846; Santa Cruz, California). -Actin was probed as a loading control. Then membranes were washed and incubated with horseradish peroxidase–conjugated secondary antibody (sc-2004 or sc-2005; Santa Cruz, California). Western blot analysis was performed using the Odyssey Infrared Imaging System (Li-Cor, Lincoln, Nebraska).

Total lysates from different treated MEPM cells using 5× SDS-lysis buffer supplemented with proteases inhibitors (M250, Amresco, Ohio) were determined. Protein concentration was determined using a standard BSA protein assay (Dingguo, Beijing, China). A total of 40 μg proteins were fractionated on 12% SDS-PAGE, and transferred to nitrocellulose membranes. After blocking with 5% nonfat milk, the membranes were immunoblotted with the primary antibodies: Smad2 (ab71109, Abcam, Massachusetts), p-Smad2 (sc101801, Santa Cruz, California), Smad3 (BM3559, Boster Biotech, Wuhan, China), p-Smad3 (GD-CZ5616R, Santa Cruz), Smad4 (PB0446, Boster Biotech), and Smad7 (sc-365846, Santa Cruz). β-actin was probed as a loading control. Then membranes were washed and incubated with HRP-conjugated secondary antibody (sc-2004 or sc-2005, Santa Cruz), Western blot analysis was performed using the Odyssey Infrared Imaging System (Li-Cor Lincoln, Nebraska).