Anti p smad1

Proteins were extracted by radioimmunoprecipitation assay (RIPA) Lysis buffer (Abcam, Cambridge, MA, USA) and quantified by bicinchoninic acid (BCA) Protein Assay Kit (Thermo Scientific, Rockford, IL, USA). 40 µg of protein per sample was separated onto 10% SDS-PAGE gels and transferred onto polyvinyl difluoride (PVDF) filter membrane (Bio-Rad, Hercules, CA, USA). Then, PVDF membranes were blocked with 5% non-fat dried milk for 2 h, and incubated with anti-BZW1 (1: 1000, ABcam Corp, USA), anti-Smad1 (1: 1000, ABcam Corp, USA), anti-p-Smad1 (1: 1000, ABcam Corp, USA), anti-Smad3 (1: 1000, ABcam Corp, USA), anti-p-Smad3 (1: 1000, ABcam Corp, USA), anti-TGF-β1 (1: 2000, ABcam Corp, USA) and anti-GAPDH (1: 2000, ABcam Corp, USA), respectively at 4 overnight. Finally, membranes were incubated with secondary antibodies (horseradish peroxidase-conjugated anti-rabbit). Signals were developed using an enhanced chemiluminescence reaction kit (Applygen Technologies, Beijing, China).