Cells ready for Western blotting analysis were harvested and washed with cold PBS twice, then lysed on ice in RIPA buffer (1 × PBS, 1% NP-40, 0.1% sodium dodecylsulfate (SDS), 5 mM EDTA, 0.5% sodium deoxycholate, and 1 mM sodium orthovanadate) that contained 100 μg/mL phenylmethylsul-fonylsuoride and protease inhibitors (KeyGen, Nanjing, China).
Approximately 50 μg of protein from each sample was separated using a 10% SDS-polyacrylamide gel, electrotransfered to polyvinylidene fluoride(PVDF) membranes and blocked in 5% nonfat dry milk in Tris-buffered saline, pH 7.5 (100 mM NaCl, 50 mM Tris, and 0.1% Tween-20). The transferred membranes were incubated with anti-SOX2 (Cell Signaling Technology, USA) and anti-β-actin primary antibodies (Beyotime, Jiangsu, China) overnight at 4°C, followed by incubation with horseradish peroxidase(HRP) conjugated IgG(JacksonImmunoResearch, USA). Proteins were detected by Quantity-one software (Bio-Rad, Laboratories, Inc, USA) using Immobilon ECL Chemiluminescence HRP Substrate (Millipore, Merck, USA).
Approximately 50 μg of protein from each sample was separated using a 10% SDS-polyacrylamide gel, electrotransfered to polyvinylidene fluoride(PVDF) membranes and blocked in 5% nonfat dry milk in Tris-buffered saline, pH 7.5 (100 mM NaCl, 50 mM Tris, and 0.1% Tween-20). The transferred membranes were incubated with anti-SOX2 (Cell Signaling Technology, USA) and anti-β-actin primary antibodies (Beyotime, Jiangsu, China) overnight at 4°C, followed by incubation with horseradish peroxidase(HRP) conjugated IgG(JacksonImmunoResearch, USA). Proteins were detected by Quantity-one software (Bio-Rad, Laboratories, Inc, USA) using Immobilon ECL Chemiluminescence HRP Substrate (Millipore, Merck, USA).