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. 2000 Sep 5;192(5):719–728. doi: 10.1084/jem.192.5.719

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© 2000 The Rockefeller University Press

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).

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graphic file with name JEM000900.f5a.jpg — Flt3 preferentially activates Stat5a in COS7 cells. (A) COS-7 cells were transfected with Stat5a, Stat5b, Flt3, or control vector in the combinations shown. After stimulation with either medium control or FL, cells were lysed and Stat5a or Stat5b was immunoprecipitated from cell lysates and immunoblotted with antiphosphotyrosine antibody. The same membrane was stripped and reblotted with anti-Stat5 antibody that recognizes both Stat5a and Stat5b. The expression of Flt3 was confirmed by immunoblot with anti-Flt3 antibody on total cell lysates. (B) Nuclear extracts from COS7 cells transfected with Stat5a, Stat5b, Flt3, or vector control in the combination shown were incubated with ³²P-labeled probe and subjected to EMSA. Oligonucleotide competition was performed by preincubating nuclear extracts with the cold probe. For supershifts, nuclear extracts were preincubated with the anti-Stat5a or -Stat5b antibodies. The arrow indicates the DNA–Stat5 complex. (C) COS-7 cells were transfected with Stat5a plus control vector, Flt3, Flt3-ΔCT, or Flt3-ΔCT/TKII, as shown. The tyrosine phosphorylation of Stat5a was examined by immunoprecipitation and immunoblotting with antiphosphotyrosine antibody. The same membrane was stripped and reblotted with anti-Stat5a antibody. pY, antiphosphotyrosine antibody; SS, supershift.

graphic file with name JEM000900.f5b.jpg — Flt3 preferentially activates Stat5a in COS7 cells. (A) COS-7 cells were transfected with Stat5a, Stat5b, Flt3, or control vector in the combinations shown. After stimulation with either medium control or FL, cells were lysed and Stat5a or Stat5b was immunoprecipitated from cell lysates and immunoblotted with antiphosphotyrosine antibody. The same membrane was stripped and reblotted with anti-Stat5 antibody that recognizes both Stat5a and Stat5b. The expression of Flt3 was confirmed by immunoblot with anti-Flt3 antibody on total cell lysates. (B) Nuclear extracts from COS7 cells transfected with Stat5a, Stat5b, Flt3, or vector control in the combination shown were incubated with ³²P-labeled probe and subjected to EMSA. Oligonucleotide competition was performed by preincubating nuclear extracts with the cold probe. For supershifts, nuclear extracts were preincubated with the anti-Stat5a or -Stat5b antibodies. The arrow indicates the DNA–Stat5 complex. (C) COS-7 cells were transfected with Stat5a plus control vector, Flt3, Flt3-ΔCT, or Flt3-ΔCT/TKII, as shown. The tyrosine phosphorylation of Stat5a was examined by immunoprecipitation and immunoblotting with antiphosphotyrosine antibody. The same membrane was stripped and reblotted with anti-Stat5a antibody. pY, antiphosphotyrosine antibody; SS, supershift.

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