4.10. Western Blot Analysis

The cells were lysed using RIPA buffer, supplemented with protease and phosphatase inhibitors (ATTO, Tokyo, Japan). The concentrations of the proteins were determined using the Pierce^® BCA Protein Assay Kit (Thermo Fisher Scientific, Inc., Rockford, IL, USA). Equal amounts of cell lysates were separated by 7.5–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to polyvinylidene difluoride (PVDF) membranes (EMD Millipore, Hayward, CA, USA) using standard electroblotting procedures. The blots were blocked with 5% skim milk in Tris-buffered saline with Tween-20 (TBST) at room temperature for 1 h, and immunolabeled with the primary antibodies against cleaved capase-9, cleaved caspase-3, PARP, p21, p53, phospho-ERK1/2, ERK1/2, phospho-AKT, AKT, phospho-STAT3, STAT3, ALDH1A1, integrin α6, Sox2, Oct4, Nanog, CD133, α-tubulin (dilution 1:2000), and β-actin (dilution 1:10000) by incubating overnight at 4 °C. After washing thrice with TBST, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse (dilution 1:3000) secondary antibody for 1 h at room temperature. Immunolabeling was detected using an enhanced chemiluminescence (ECL) kit (Bio-Rad Laboratories, Hercules, CA, USA), according to the manufacturer’s instructions. The density of the bands was analyzed using ImageJ software, version 1.5 (NIH).