STAT3 activation by G-CSF-R tyrosine mutants. (A) STAT3 immunoprecipitation from lysates of Ba/F3 cells expressing WT or mutant G-CSF-R proteins, as described in Fig 2B. Multiple analyses of at least three independent clones of each mutant gave equivalent results. (B) EMSA of nuclear extracts from Ba/F3 cells expressing WT G-CSF-R (WT) or mutants, as described in Fig 2A, at the concentrations of G-CSF shown. (C) EMSA of serially diluted nuclear extracts from Ba/F3 cells expressing WT or mA receptors stimulated with 100 ng/mL G-CSF. Extracts from the equivalent of 4, 2, 1, and 0.5 × 10⁵ cells were used. (D) Quantitative analysis of EMSA shown in (C), setting STAT3 binding from 4 × 10⁵ Ba/F3[WT] cells at 100%. Results from dilution of Ba/F3[WT] extracts are shown with filled squares, and from Ba/F3[mA] extracts with open diamonds.