Immobilon western chemiluminescent horseradish peroxidase hrp substrate kit

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).

The Western Blot analysis was performed as described previously [31 (link)]. To determine the alterations of protein levels, 40 µg of total protein were separated by 12% and by 7.5% denaturing SDS-PAGE and transferred onto 0.2 µm nitrocellulose membranes overnight. The membranes were blocked with a 5% milk powder solution for 1 h and incubated with primary anti-PARP-IgG (1:1.000, Cell Signaling, #9532), anti-H2AX-IgG (1:10.000, Millipore, #07-627), anti-γH2AX-IgG1 (1:5.000, Millipore, #05-636), anti-cyclin-B1 (1:5.000, Santa Cruz, #sc-752), or anti-phospho-H3 (1:1.000, Cell Signaling, #9701) at 4 °C overnight. The membranes were then incubated with secondary horseradish-peroxidase-coupled antibodies goat-anti-mouse IgG (H+L) (1:10.000, Thermo Fisher Scientific, #A-11029) or goat-anti-rabbit IgG (H+L) (1:10.000, Thermo Fisher Scientific, #31460) for 1 h at rt. GAPDH served as a control for equal loading of the samples, the blots were incubated with HRP-labeled anti-GAPDH (1:100.000, Abnova, #MAB5476) for 45 min at rt. The blots were developed using the Immobilon Western Chemiluminescent horseradish peroxidase (HRP) substrate kit (Merck Millipore). Images were captured using a GeneGnome (Syngene).