Characterization of STAT3/G-CSF-R interactions. (A) Competition of STAT3 containing EMSA complexes with phosphopeptides specific for each of the cytoplasmic tyrosines of the murine G-CSF-R receptor. Nuclear extracts from Ba/F3[WT] cells stimulated with G-CSF for 10 minutes were incubated either without peptide (−) or with the indicated phosphopeptides at a concentration of 500 μmol/L for 60 minutes, followed by EMSA. (B) Peptide competition as described in (A), with no peptide (−) or with Y703 and Y743 peptides at concentrations of 500, 200, and 50 μmol/L. (C) Direct binding of the STAT3 SH2 domain to tyrosine-phosphorylated G-CSF-R in vitro. Purified GST proteins immobilized on glutathione-Sepharose beads were incubated with purified, tyrosine-phosphorylated G-CSF-R cytoplasmic domain and washed extensively. Bound G-CSF-R was determined by boiling the beads in SDS sample buffer and subjecting the supernatant, along with the input G-CSF-R, to Western blot analysis with anti–G-CSF-R (–G-CSF-R) or anti-phosphotyrosine (-pY) antibodies, as indicated.