P bad

SDS-Page electrophoresis and transfer of proteins to nitrocellulose membranes were performed according to standard procedures. Ponceau S (0.1%) staining of membranes verified transfer of proteins and equal loading. Membranes were incubated with antibodies to CD133 (Miltenyi Biotec, Auburn, CA, USA), cleaved and intact PARP, BAD, p-BAD, BCL-XL, MCL-1 (BioLegend, San Diego, CA, USA) cleaved active caspase-3, cleaved active caspase-9, BCL-2, active BAX (Novus Biologicals, Centennial, CO), AKT and p-AKT (Santa Cruz Biotech, Dallas, TX, USA), ERK, p-ERK, or to β-Actin (ProteinTech, Rosemont, IL, USA) as a loading control. Immunoblots were sequentially reprobed with other antibodies after stripping them of antibodies. Immune complexes were detected by incubation with horseradish peroxidase-conjugated antibodies to mouse or rabbit IgG (1:3000), followed by enhanced chemiluminescence (ECL; Pierce, Rockford, IL, USA) and imaging in a GE Healthcare Amersham Imager 600.

Total cell lysates were subjected to SDS-Page electrophoresis, and proteins were transferred to nitrocellulose membranes according to standard procedures. To verify the transfer of proteins and equal loading, membranes were stained with Ponceau S (0.1%). Membranes were then incubated with antibodies to CD133 (Miltenyi Biotec, Auburn, CA, USA), AREG (Santa Cruz Biotech, Dallas, TX, USA), p-EGFR, p-MEK, p-BAD (BioLegend, San Diego, CA, USA), p-ERK, total MEK, ERK, BAD, proliferating cell nuclear antigen (PCNA), cyclin D1, or β-actin (ProteinTech, Rosemont, IL, USA) as loading control. After stripping membranes of antibodies, immunoblots were sequentially re-probed with other antibodies, and immune complexes were detected by incubation with horseradish peroxidase-conjugated antibodies to mouse or rabbit IgG (1:3000), followed by enhanced chemiluminescence (ECL; Pierce, Rockford, IL, USA) and imaging in a GE Healthcare Amersham Imager 600.