Anti rabbit or anti mouse igg horseradish peroxidase

For Western blot analysis, 1.25×10⁶ NSCs were cultured in 2.5 mL media unless specified otherwise. Proteins in cell lysates (20 μg protein) were separated via sodium dodecyl sulfate–polyacrylamide gel electrophoresis and electroblotted onto a polyvinylidene difluoride (PVDF) membrane for 90 min at 100 V at 4 °C. The membranes were blocked with 5% BSA for 60 min in TBST (20 mM Tris–HCl, pH 7.5, 50 mM NaCl, 0.1% (vol/vol) Tween 20), and incubated overnight at 4°C with primary antibodies diluted (1:1000) in TBST. After being washed in TBST buffer, the membranes were incubated for 60 min in anti-rabbit or anti-mouse IgG–horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA, USA) secondary antibodies diluted (1:2000) in TBST, and the labeled proteins were detected with chemiluminescence reagents (Thermo Fisher Scientific, Rockford, IL, USA). Experiments were repeated three times. Western blots intensities of bands were quantitated using Image J software (NIH, Bethesda, MD) as previously described (Kwon et al., 2011 (link)).