Mouse anti human zeb1 monoclonal antibody sc 81428

Ice-cold radioimmunoprecipitation assay buffer (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was adopted to isolate total protein from tissues or cells. The total protein concentration was examined using a BCA assay kit (Santa Cruz Biotechnology). Equal quantities of total protein were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto a polyvinylidene difluoride membrane (EMD Millipore, Billerica, MA, USA), and then blocked in TBS/0.1% Tween (TBST) containing 5% milk at room temperature for 2 h. The membranes were incubated at 4°C overnight with primary antibodies: mouse anti-human ZEB1 monoclonal antibody (sc-81428; 1:1,000 dilution; Santa Cruz Biotechnology) or mouse anti-human GAPDH monoclonal antibody (sc-137179; 1:1,000 dilution; Santa Cruz Biotechnology). After being washed with TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (sc-2005; 1:5,000 dilution; Santa Cruz Biotechnology) at room temperature for 2 h. Finally, an enhanced chemiluminescence reagent (Bio-Rad Laboratories, Hercules, CA, USA) was used to detect protein signals. GAPDH was used as internal control.