Characterization of the Jab1-containing complexes in hematopoietic cell lines. (A) Equal amounts of cell lysate from CML cell lines (K562, KYO-1, MEG-01, and MOLM1) and non-CML cell lines (CMK and HEL) were analyzed by immunoblotting using antibodies against Jab1, p27, Abl, and β-actin. (B) Lysate from K562 was fractionated through a 10% to 40% glycerol gradient by centrifugation at 27 000 rpm for 24 hours at 4°C. Each fraction was analyzed by immunoblotting using antibody against Jab1 (first row). Direct lysates (second row, left) or the same fractions used in the upper panels (second row, right) were separated by nondenaturing gel electrophoresis (native-PAGE) and analyzed by immunoblotting using antibody against Jab1. (C) Cell lysates extracted from several CML cell lines and primary cells from patients with CML in chronic phase (CML-CP) and accelerated phase (CML-AP) as well as non-CML cell lines and other primary cells were separated by native-PAGE and analyzed by immunoblotting using antibody against Jab1. (D) K562 cells were electroporated with a mock vector and an HA-tagged Jab1 expression vector together with a neo marker–containing plasmid. After G418 selection, cell lysates were separated by SDS-PAGE (left panels) and native-PAGE (right panels), and analyzed by immunoblotting using antibodies against Jab1, HA-epitope, p27, γ-tubulin, and Cul1. The cell-cycle distributions of the GFP-positive cells were analyzed using a flow cytometer after staining with Hoechst 33342. The averages of 3 independent experiments and standard deviations are shown.