scribed. For camptothecin treatment, cells were incubated for 24 hours in the presence of 0.1 or 1 M camptothecin, followed by harvesting and Western analysis as described below. PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19723632 Cell pellets Frozen cell pellets from the NCI 60-cell line panel were obtained from the Division of Cancer Treatment and Diagnosis Tumor repository of the National Cancer Institute and were stored at -80C until use. The cell lines for which cell pellets 
were received are listed in. Tissue K-858 chemical information specimens All tissue specimens used in this study were frozen, anonymous, publicly available archival specimens of non-small cell lung cancer with paired non-malignant control tissue, or specimens from benign lung tumors. All specimens were received from the Western Division of the Cooperative Human Tissue Network, Vanderbilt University, Nashville, TN. No identifiable private patient or donor information was supplied with the specimens. Since no data was obtained through intervention or interaction with the individual and since no identifiable private information was supplied to us, this research did not constitute human subjects research as defined by CFR 46.102f and the OHRP Guidance on Research Involving Coded Private Information or Biological Specimens, and was granted exemption from review by the Torrey Pines Institute for Molecular Studies IRB. Western analyses Cell pellets or two near-confluent 10-cm plates of PBS-washed H358 cells were lysed by the addition of 350 l per pellet or 700 l per plate of cold RIPA buffer to which complete protease and phosphatase inhibitors were added just prior to use, and processed as PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19723701 previously described. Frozen tissue specimens were homogenized in 3 ml of RIPA buffer on ice using a tissue grinder and then centrifuged to remove debris. The cell and tissue lysates were aliquoted and frozen at -80C until use. A freshly thawed lysate was used for each gel run. Lysates were resolved by SDS-PAGE using Criterion 1020% Tris-HCl precast gels. An equivalent amount of a freshly thawed aliquot of the reference stock of H358 lysate was included in each run as a common control. Following electrophoreses, proteins were transferred to PVDF membranes and immunostained by incubation with primary antibody, followed by appropriate HRP-conjugated secondary antibody, and Pierce ECL reagent, followed by exposure to film. Films were scanned using an Alpha Imager and bands were quantified using the accompanying software. PS506 band intensities were normalized to the reference H358 control. Immunoprecipitation/Western blotting Immunoprecipitations were carried out essentially as described. Briefly, cell lysates from exponentially growing H358 cells were prepared in RIPA buffer as described above. Cellular proteins were immunoprecipitated by rocking overnight with 50 l goat anti-topo I. Immunocomplexes were collected by the addition of 20 l protein AG agarose followed by centrifugation. Immune complexes were dissociated at low pH in the absence of dithiothreitol or -mercaptoethanol to avoid dissociation of the IgG. Electrophoresis sample buffer was added and samples were subjected to SDS-PAGE as described for Westerns, without prior boiling to further ensure that the IgG remained intact. The Western was processed as described above except that Clean-Blot IP Detection Reagent was used to detect the primary antibody. ELISA analysis Serine 506-phosphorylated and non-phosphorylated forms of the 21-amino acid topo I peptide described above were synthesized by Biopeptid