EMSA supershift analysis and DNA-affinity chromatography of BAF/BO3 cells transfected with full-length and truncated mutants of G-CSFR. BAF/BO3 cells were stably transfected with vector containing either wild-type human G-CSFR (HRG-183) or with vector containing G-CSFR constructs truncated at intracytoplasmic amino acid residue 96 (HGR-96), residue 57 (HGR-57) or residue 26 (HGR-26) as described.36 The constructs contained one or more of the homology regions, boxes 1 and 2, and tyrosines (Y) as indicated in panel A. Cells were extracted before (−) or after (+) G-CSF or interferon-γ stimulation. EMSA was performed without (−) or with (+) antibody to Stat3β. The position of the SIF-A, B, and C complexes and the supershifted(SS) Stat3β complex are indicated on the right. The results shown are each representative of two experiments. In (B), the stably transfected cell lines (B-26, B-57, B-96, and B-183) and the parental cell line (BO3) were stimulated with G-CSF (100 ng/mL for 15 minutes) and extracted. Whole-cell protein extracts (500 μg each) were bound to and eluted from a hSIE-affinity purification column. Eluted proteins were separated by SDS-PAGE, immunoblotted with Stat3 monoclonal antibody, and developed with ECL chemiluminescence. The position of the prestained molecular-weight markers are shown on the left and the position of the Stat and Stat3β bands are shown on the right. In (C), whole-cell protein extracts of each cell line (50 μg each) were separated by SDS-PAGE and immunoblotted with Stat3 monoclonal antibody. The positions of Stat3 and Stat3β are indicated on the right.