Ectopic expression of Bcr-Abl induced the small Jab1 complex. (A) TonB cells, able to express Bcr-Abl on addition of tetracycline or its analog doxycycline, and parental cells, BaF3, were cultured with and without doxycycline in the absence of IL-3 for 24 hours. After 24 hours of incubation, cell lysates were separated by native-PAGE (first and second rows) and SDS-PAGE (third to sixth rows), and analyzed by immunoblotting using antibodies against Jab1, γ-tubulin, p27, and Abl. The relative amounts of the small Jab1 complex and p27 are presented as the ratio of the small Jab1 complex–γ-tubulin and p27–γ-tubulin and calculated with the level of untreated BaF3 cells as 1.0. (B) Murine bone marrow cells were infected with the MSCV-p210Bcr-Abl-IRES-GFP retrovirus and plated in liquid culture. Transformed bone marrow cells were cultured with and without STI571 for 8 hours. After 8 hours of treatment, cell lysate was separated by native-PAGE (first and second rows) and SDS-PAGE (third to fifth rows), and analyzed by immunoblotting using antibodies against Jab1, γ-tubulin, and p27. The relative amounts of the small Jab1 complex and p27 are presented as the ratio of the small Jab1 complex–γ-tubulin and p27–γ-tubulin and calculated with the level of untreated bone marrow cells expressing Bcr-Abl as 1.0. BM indicates bone marrow cells. (C) BaF3 cells, which were routinely maintained in IL-3, were cultured in the absence of IL-3. At the indicated times, cells were harvested and the cell lysates were separated by native-PAGE (first and second rows) and SDS-PAGE (third to fifth rows), and analyzed by immunoblotting using antibodies against Jab1, γ-tubulin, and p27. The relative amounts of the small Jab1 complex and p27 are presented as the ratio of the small Jab1 complex–γ-tubulin and p27–γ-tubulin and calculated with the level of untreated BaF3 cells as 1.0.