Analysis of transforming properties and imatinib sensitivity of EML1-ABL1. (A) Ba/F3 cells retrovirally transduced with indicated constructs were grown in the absence or presence of IL3. Their mean growth ± SD was recorded over a period of 3 days. (B) Western blot analysis of retroviral-transduced Ba/F3 cells. Constitutive activation of EML1-ABL1 and BCR-ABL1 kinases is shown by immunoblotting with anti-phospho-ABL1 (anti-p-ABL1). Expression of the 3 ABL1 fusion proteins is demonstrated using an anti-ABL1 antibody. Activation of Erk1/2 and Stat5 is demonstrated using anti-phospho-ERK1/2 and anti-phospho-STAT5 antibodies. (C) EML1-ABL1- and BCR-ABL1-transduced Ba/F3 cells were treated with the indicated concentrations of imatinib and cell survival was quantified after 24 hours. Cell survival in the absence of imatinib (= control) was set at 100%; the results represent the average ± SEM of 3 determinations. (D, top panel) Western blot showing the effect of imatinib treatment on EML1-ABL1-expressing Ba/F3 cells. Total cell lysates were analyzed using antiphosphotyrosine (4G10) antibody, indicating a dose-dependent decrease in phosphorylation of EML1-ABL1, Stat5, and Lyn upon imatinib treatment. (D, bottom panel) Decrease of Lyn activity upon imatinib treatment was confirmed by immunoprecipitation of Lyn followed by detection of its phosphorylation on Tyr396 with anti-phospho-SRC. The blot was stripped and reprobed with anti-LYN.