additional phosphatase inhibitor mixture was added in to RIPA protease inhibitor combination. Protein concentration was measured by BCA protein assay kit. Equal levels of cell lysates were subjected to SDS PAGE, used in NC filters, ATP-competitive Aurora Kinase inhibitor and probed with the antibody for protein recognition. For Ip Address analysis, equal levels of cell lysate were first incubated with the anti HA antibody for 1 hour and, subsequently, reacted with protein A/G conjugated beads over night at 4 C or directly incubated with the anti ALK antibody conjugated beads. The pulled-down beads were washed and exposed to Western blot analysis for protein detection. Immunohistochemistry IHC assays were done on six human lung cancer tissue sections with ALK mutations, four human lung cancer erythropoetin sections without ALK mutations, two normal human lung sections from Pantomics, five human lung cancer tissue arrays containing 37 normal lung sections and 263 lung cancer sections from Pantomics, three human tissue arrays from US Biomax including ALCL, rhabdomyosarcoma, and normal lymph node, and OCT stuck frozen growth sections prepared from the xenografted nude mice. After deparaffinization, all sections were treated with a few months H2O2 buffer for thirty minutes to inactivate the endogenous peroxidase activities and then incubated in 0. 01 M sodium citrate buffer for antigen retrieval. After stopping with 10% normal goat serum, these sections were reacted with mentioned antibodies at 4 C for overnight. Consequently, these sections were incubated with diaminobenzidine discoloration, HRP plastic conjugate, and then Mayer hematoxylin. cells were seeded into the cell migration insert containing 350 ul of Dulbecco modified Eagle medium and then placed into the well containing 750 ul of 10% fetal pifithrin alpha bovine serum/Dulbecco modified Eagle medium in a 24 well plate. After 18 hours of incubation, transferred cells were fixed with hundreds of methanol and stained with Giemsa solution. The amount of transferred cells was measured by the Image Pro Plus research program. The amount of colonies formed was measured from the Image Pro Plus research system. In Vitro Kinase Assay In vitro ALK activity of H1299 transfectants was measured by common tyrosine kinase assay kit. In short, cells were first lysed in lysis buffer. After quantifying the protein concentration using the BCA assay, equal levels of cell lysates were immunoprecipitated using the anti HA antibody, and the ALK precipitated complex was then added into the wells coated with poly Glu Tyr substrate. After 30-minutes of incubation, the peroxidase conjugated antiphosphotyrosine antibody was added to the wells. After incubating with the Horseradish peroxidase substrate alternative, the wells were read in an ELISA reader set at an absorbance of 450 nm. Immunofluorescence After the cells were fixed in four or five formaldehyde/phosphate buffered saline and permeabilized in 0.