Horseradish peroxidase conjugated goat anti mouse secondary antibody sc 2005

Protein was isolated using radioimmunoprecipitation assay lysis buffer (Beyotime, Shanghai, P.R. China) from tissue samples or cells. A bicinchoninic acid protein assay kit (Beyotime) was used to detect the protein concentration. Equal amounts of protein were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were then blocked at room temperature for 1 h with Tris-buffered saline with Tween 20 (TBST) containing 5% nonfat milk and incubated with primary antibodies overnight at 4°C. Subsequent to washing thrice with TBST, the membranes were further incubated with horseradish peroxidase-conjugated goat anti-mouse secondary antibody (sc-2005; 1:5,000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at room temperature for 2 h. We visualized the protein blots using an enhanced chemiluminescence detection system (Pierce, Rockford, IL, USA) and analyzed the band intensities with Quantity One software version 4.62 (Bio-Rad Laboratories, Inc.). The primary antibodies used in this study included mouse anti-human ZEB2 monoclonal antibody (sc-271984; 1:1,000 dilution; Santa Cruz Biotechnology) and mouse anti-human GAPDH (sc-47724; 1:1,000 dilution; Santa Cruz Biotechnology) antibody.