Activation of STAT5 DNA binding and STAT-dependent Py origin replication in BA/F3 cells through a series of mutants of hGM-CSFR.

(A) BA/F3 cells expressing wild-type α subunit and various β mutants were depleted of mIL-3 for 5 hours and restimulated with hGM-CSF (10 ng/mL) for 15 minutes. Gel shift assay was done using an oligonucleotide corresponding to the STAT binding site. Arrow indicates specific STAT binding complex to the probe. (B) Plasmid containing 4 tandem repeats of STAT5 binding sites or mutant STAT5 binding sites fused to Py virus replication origin were transfected to Ba/F-wild cells and left for 5 hours in depletion media. Cells were restimulated with hGM-CSF (10 ng/mL) and harvested after 24 hours of incubation, followed by replication assay. Transfected plasmids were extracted using Hirt solution and digested with DpnI, which selectively degrades an unreplicated plasmid, and then the plasmids were separated through an agarose gel and analyzed by Southern blotting. (C) Plasmid containing wild-type STAT5 binding site followed by Py virus replication origin was transfected to BA/F3 cells expressing various hGM-CSFR mutants, and replication induced by hGM-CSF was analyzed as described above. Arrow indicatesDpnI-resistant replicated bands.