Two-dimensional gel analysis of proteins from K562 cells. (A) Protein profiles of extracts from K562 cells incubated with DMSO (left) or with imatinib (right) for 24 hours. The marked protein spots were chosen for further characterization by MALDI-MS and ESI-MS/MS because they were expressed to elevated relative levels either in control cells (left) or in cells after imatinib treatment (right). (B) Immunoblots of some representative proteins to confirm the results of 2-D gels. Expression of vinculin and Ran-binding protein was down-regulated, and expression of β-tubulin was up-regulated in K562 cells after incubation with imatinib for 24 and 48 hours. Normalization was performed using α-tubulin. (C) Enlarged regions of Coomassie-stained 2-D gels. Arrows indicate the location of eIF5A. (D) RT-PCR for the detection of eIF5A expression in K562, HL-60, and BA/F3p210, including the mutants M351T, T315I, and E255K as well as wt-BA/F3 cells. Cells were incubated with imatinib for 24 hours and compared with untreated control. (E) RT-PCR for the detection of eIF5A mRNA levels in PBMCs from 2 healthy donors and 7 CML patients. Normalization was performed with human 18S rRNA.