The phosphotyrosine pY245 and pY412 c Abl antibodies were from Cell Signaling Technology, Beverly, MA, USA. The h Abl K12 antibody was obtained from Santa Cruz Biotechnology, Santa Cruz, CA, USA. Anti phosphotyrosine 4G10 antibody was obtained from Upstate Biotechnology, Lake Placid, NY, USA. Horseradish peroxidase conjugated antibodies and the ECL detection program were from Amersham Pharmacia Biotech, Uppsala, Sweden. Tunicamycin and cisplatin diamine dichloride were from Sigma, St. Louis, MO, USA.. COS cells and btc 6 cells were preserved in DMEM supplemented with 10 % fetal calf serum, benzylpenicillin 100 U/ml and streptomycin Gefitinib structure at 3-7 C and five hundred CO2. COS cells were maintained as described above in 5 cm culture dishes, washed three times in serum free medium and transfected with 1 ug of each plasmid or empty vector, using 1-4 ul Lipofectamine. The plasmid containing wild typ-e human Shb cDNA inserted within the pcDNA1 vector has been described previously. The Quickchange XL site directed mutagenesis kit was employed to execute site directed mutagenesis of Shb at tyrosines 333, 355, 384 and 423. Metastasis The mutants created were confirmed by DNA sequencing. The Shb SH2 GST fusion protein plasmid was described previously. The Shb PTB Pro GST plasmid was described previously as p55 ShbSH2 and encodes a protein containing both proline rich sequences and the PTB domain. The vector encoding human wild type d Abl, was a present from Philippe Soubeyran. Kinase lazy c Abl in pcDNA3 was generously given by Dr Ann M. Pendergast, Durham, NC, GST fusion protein plasmids corresponding to the c Abl SH3 domain and c Abl SH2 SH3 domains were a-kind gift from Bruce J Mayer. COS cells were often left untreated or treated with pervanadate for 15 min at 37 C, after that the cells were washed three times with ice cold PBS and subsequently lysed in lysis buffer on ice for 10 min. Nuclei were pelleted by centrifugation and extracts were incubated with either Shb or d Abl rabbit polyclonal antibodies. Immune complexes were pelleted with 50 ul Protein A Sepharose and washed 3 times in PBS, week or two Triton X 100 and once with H2O. Samples were then resolved by SDS PAGE and transferred onto Immobilon filters in 20% order JNJ 1661010 methanol, 190 mM glycine, 2-3 mM Tris and 0. 02% SDS. The blots were blocked in PBS, five full minutes BSA, 0. Five hundred Tween 20 and incubated with main antibodies as indicated. Immunoreactivity was detected using horseradish peroxidase conjugated secondary antibodies and ECL. Cell extracts from COS cells transiently overexpressing wild type Shb or Shb with one tyrosine residuemutated or d Ablwere put into aliquots of GST tagged fusion proteins, immobilized on glutathione Sepharose beads. The samples were incubated, washed and resolved on SDS PAGE as described above. The cells had been pre treatedwith Calpain Inhibitor II andsomegroups also with pervanadate before lysis.