Horseradish peroxidase conjugated goat anti mouse igg secondary antibody sc 516102

The extraction of total protein from cultured cells was performed with RIPA lysis buffer (Solarbio, Beijing, China). The concentration of total protein was measured with the BCA Protein Assay Kit (Beyotime, Shanghai, China). The equivalent amounts of total protein were separated by SDS polyacrylamide gel electrophoresis in a 10% gel and transferred to polyvinylidene difluoride membranes, which were then blocked at room temperature for 2 h in 5% fat-free milk diluted in Tris-buffered saline with 0.1% of Tween 20 (TBST). After overnight incubation at 4°C with primary antibodies in TBST, the membranes were washed with TBST thrice and then incubated with a horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (sc-516102; 1:5000 dilution; Santa Cruz Biotechnology, Dallas, TX, USA) at room temperature for 2 h. The detection of protein signals was carried out by means of an Enhanced Chemiluminescence Detection System (Pierce, Rockford, IL, USA). The primary antibodies used in this study included a mouse anti-human ETS1 monoclonal antibody (sc-55581; 1:1000 dilution; Santa Cruz Biotechnology), and a mouse anti-human GAPDH antibody (sc-47724; 1:1000 dilution; Santa Cruz Biotechnology). GAPDH served as a loading control.