Glyceraldehyde 3 phosphate dehydrogenase gapdh

Total BON cell lysates following dacarbazine ± ABT-888 treatment were prepared and analyzed by Western blotting as previously described^{44 (link)}. Each antibody was diluted as follows: 1:2000 for mammalian achaete-scute complex-like1 (ASCL1) (BD Pharmingen, San Diego, CA, USA), 1:3000 for chromogranin A (Zymed Laboratories, San Francisco, CA, USA), 1:10,000 for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Trevigen, Gaithersburg, MD, USA), and 1:1000 for p21^Waf1/Cip1, cleaved poly (ADP-ribose) polymerase (PARP), phosphorylated ATM, total ATM, and Survivin (Cell Signaling Technology, Beverly, MA, USA). Antibody signals were detected using Supersignal West Femto, Dura, or Pico (Pierce, Rockford, IL, USA) chemiluminescence systems and manufacturers’ instructions were adhered to.

FTC-236 and HTh7 cells were transfected transiently with EBNA2, Notch1, or an empty vector control (pSG5 or LNCX1). After 48 h, cells were washed with PBS and then treated with lysis buffer. The protein lysates were prepared as described previously.^{11 (link)} Protein concentration was quantified using the BCA Protein Assay Kit (Thermo Scientific, Waltham, MA) according to the manufacturer’s instructions. Denatured protein extracts were resolved by electrophoresis on 4% to 15% Criterion TGX precast gels (Bio-Rad Laboratories, Hercules, CA), transferred onto nitrocellulose membranes (Bio-Rad Laboratories), then blocked and incubated with the following primary antibodies overnight at 4°C: β-actin (1:2,000; Cell Signaling Technology, Danvers, MA), Notch1 (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:10,000; Trevigen, Gaithersburg, MD), and anti-EBNA2 PE2 (1:250; Abcam, Cambridge, United Kingdom).
After washing, membranes were incubated for 1 h at room temperature with horseradish peroxidase–conjugated, antirabbit/mouse secondary antibodies. The immunoreactive bands were visualized using Immunstar (Bio-rad Laboratories), Supersignal West Pico, or SuperSignal West Femto (Pierce Biotechnology, Rockford, IL) reagents. Expression levels of β-actin or GAPDH were used as the loading control.