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IL-3 was withdrawn from NFS60 cells. A cytokine-independent variant of NFS60 grew out, which was used for transfection experiments. The G-CSF signaling characteristics of this line are similar to the parental NFS60S. This line was transfected by electroporation with the plasmid 4×TTCC1Ctkluc, which contains four copies of the STAT-binding element 5′-GATCTGCTTCCCGGAACGT-3′ cloned into the plasmid tk-luc (21), and the plasmid pSV2neo, which confers neomycin resistance. Single drug-resistant clones were isolated by limiting dilution and screened for increased luciferase expression after a 2.5-hour exposure to 10 ng/ml G-CSF.
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We thank C. Sincich and P. Sanders for technical assistance and R. Chedester for help with the manuscript.
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