Phospho h2ax

Protein extraction was performed using NP-40 buffer supplemented with protease and phosphatase inhibitors (Roche, Indianapolis, IN). Quantification was performed by Bradford. Proteins were resolved in 10% polyacrylamide gels in 1X Tris/glycine/SDS buffer and transferred onto nitrocellulose membrane at 400 milliamps for 1:45 hours in 1X Tris/glycine buffer with 20% methanol. Membranes were blocked in either 5% BSA or 5% milk in TBS buffer. Primary antibody incubation was performed overnight at 4°C in 5% BSA in TBS buffer supplemented with 1% Tween-20 (TBS-T). Membranes were washed (3X) with TBS-T buffer and incubated in 1% milk in TBS-T with secondary antibodies (Li-COR, Lincoln, NE) for 1 hour. Membranes were then washed with TBS-T and scanned using the odyssey IR scanner (Li-COR). Image capture and signal analysis was done using the Image Studio software (Li-COR). Primary antibodies used in this study were: phospho-H2AX (1:1000, Abcam, Cambridge, MA), p53 (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA) and beta-actin (1:2000, Sigma, St. Louis, MO).

Primary antibodies for immunoblot include: MYC (CST #13987, 1:1,000), MYCN (Santa Cruz sc-791, 1:200), ODC1 (EMD Millipore, MABS36, 1:100), ASCL1 (BD Bioscience, BDB556604, 1:300), phospho-4EBP1 (CST #2855, 1:1,000), 4EBP1 (CST #9644, 1:2,000), phospho-S6 (CST #2211, 1:1,000), S6 (CST #2217, 1:1,000), phospho-H2AX (CST #9718, 1:1000), PARP (CST #9532, 1:1000), ASL (Abcam#201026), ASS1 for mouse tumors (Abcam #170952, 1:1,000), ASS1 for human PDX and cell lines (Polaris Pharmaceuticals, 1:1000), and HSP90 (CST #4877, 1:1,000).