Pim-1 reduces DNA binding and tyrosine phosphorylation of STAT5. Starved FD/Neo, FD/Pim44, or FD/NT81 cells were left unstimulated or stimulated with IL-3 for 6 hours as indicated. (A) Aliquots of nuclear extracts (10 μg) prepared from cell lysates were subjected to electrophoretic mobility shift assay. Protein-DNA complexes were resolved by 4% nondenaturing PAGE and detected by autoradiography. Shown is the position of the specific STAT5-DNA complex. The relative DNA binding of STAT5 in FD/Pim44 or FD/NT81 cells as compared with FD/Neo control cells was quantitated by image analysis by measuring the intensities of specific versus nonspecific DNAcomplexes. (B) Endogenous STAT5 was immunoprecipitated (IP) from cell lysates and the samples were separated by SDS-PAGE, followed by immunoblotting with antiphosphotyrosine (PY) or anti-STAT5 antibodies. The relative phosphorylation levels of STAT5 in FD/Pim44 or FD/NT81 cells as compared with FD/Neo control cells were quantitated by image analysis by measuring the intensities of the upper (PY) versus lower (STAT5) protein bands.