Activation of signal transduction pathways by BCR/ABL proteins. (A) Levels of GTP-bound RAS in BCR/ABL-expressing cells. Pools of clones expressing the indicated vectors were labeled with [γ³²P] orthophosphate. RAS was immunoprecipitated and the proportion of GTP- and GDP-bound forms was analyzed by thin-layer chromatography. (B) PI-3k interaction with BCR/ABL proteins. (Left panel) PI-3k activity was detected in antiphosphotyrosine immunoprecipitates from a mixture of clones expressing the indicated vectors. (Right panel) p85 immunoprecipitates from the indicated mixtures of clones were analyzed after SDS-PAGE and Western blotting with anti-ABL antibody. p85 was detected with anti-p85 antibody after stripping the filter. (C) Activation of MAPK and JNK pathways by BCR/ABL proteins. (Left panel) MAPK and JNK activities were measured in the appropriate immunoprecipitates using, respectively, myelin basic protein (MBP) and GST-JUN as substrates. (Right panel) Expression of c-MYC and c-JUN mRNA was determined in total cellular RNA (10 μg) by Northern blotting. GAPDH was detected as a control for equal gel loading. (D) DNA binding activity of STAT proteins. EMSA was performed using nuclear extracts of the indicated clone mixtures and synthetic oligonucleotides containing the GAS motif as a probe. Results are representative of three to four independent experiments.