Quantification of Protein Expression by Western Blot

Western blot analysis was performed as previously described^{38 (link)}. Briefly, 30 or 40 μg of total protein were separated by 10% denaturing SDS–PAGE and transferred onto 0.45 μm nitrocellulose membranes overnight. The membranes were incubated with primary ß-Catenin (6B3) (1:1000, Cell Signaling, #9582), CD133 (1:250, Miltenyi, #130-092-395), p-AKT (Ser 473) (D9E) XP (1:1000, Cell Signaling, #4060), AKT (1:1000, Cell Signaling, #9272), p-MEK1/2 (Ser217/221) (1:1000, Cell Signaling, #9121), or MEK1/2 (1:1000, Cell Signaling, #9122) antibodies overnight at 4 °C. Secondary horseradish-peroxidase-coupled antibodies goat-anti-mouse IgG (H + L) (1:10,000,Thermo Fisher Scientific) or anti-rabbit IgG (H + L) (1:10,000, Thermo Fisher Scientific), and anti-biotin, HRP-linked antibody (1:5000, Cell Signaling) were then applied for 1 h at RT. Hybridization with GAPDH-HRP (6C5) (1:10,000–20,000, Abnova, #MAB5476) coupled antibody was performed for 30 min at RT as a housekeeping gene. Antibodies were visualized using the Immobilon Western Chemiluminescent horseradish peroxidase (HRP) substrate kit (Merck Millipore). Images were generated using Gene Genome Syngene Bio Imaging and quantified using Image J (Rasband, W.S., U.S. National Institutes of Health).