Bcr-Abl kinase regulates the small Jab1 complex in CML cells. (A) K562 cells and MEG-01 cells were treated with DMSO and STI571 (5 μM) for 8 hours. After 8 hours, cell lysates were separated by native-PAGE (first and second rows) and SDS-PAGE (third to bottom rows), and analyzed by immunoblotting using antibodies against Jab1, p27, Cul1, Skp2, phosphotyrosine (pTy), Abl, γ-tubulin, and pRb. (B) K562 cells were treated with DMSO and STI571 for 8 hours (second and third lanes). After 8 hours, STI571 was washed out and cells were resuspended in culture medium with and without cycloheximide. Lysates from cells harvested at the indicated times (fourth to seventh lanes) were separated by native-PAGE (first and second rows) and SDS-PAGE (third to sixth rows), and analyzed by immunoblotting using antibodies against Jab1, phosphotyrosine, cyclin D3, and γ-tubulin. (C) K562 cells were treated with DMSO and STI571 for 8 hours, cytocentrifuged, and fixed with 4% paraformaldehyde for 10 minutes. The fixed cells were immunostained using antibody to Jab1 (second row) and DNA was visualized with Hoechst 33342 (third row). The image was acquired using an Olympus IX 71 microscope (Olympus, Tokyo, Japan) equipped with a Cool SNAP color digital camera (Roper Scientific, Tucson, AZ). Fluorescence images were electronically recorded with Meta Com software (Universal Imaging, Downingtown, PA). Original magnification × 400. The cytoplasmic boundaries are shown with arrows. P.C. indicates phase-contrast. (D) K562 and CMK cells were treated with DMSO and STI571 for 8 hours. Cells were harvested and stained with PI, and the cell-cycle profile was analyzed using a flow cytometer. Representative histograms for each cell and the distribution of the cells are shown.