STAT3 activation in the absence of receptor tyrosines. (A) EMSA of nuclear extracts from Ba/F3 cells expressing WT G-CSF-R or mutants. Growth factor–deprived cells were incubated for 10 minutes at 37°C without factor (−) or with 100 ng/mL G-CSF (+). Nuclear extracts were prepared and incubated with32P-labeled double-stranded m67 oligonucleotide. (B) STAT3 immunoprecipitation from lysates from Ba/F3 cells expressing WT or mutant G-CSF-R proteins. Serum- and growth factor–starved cells were incubated for 10 minutes at 37°C without factor (−) or with 100 ng/mL G-CSF (+). The Western blot was hybridized with anti-phosphotyrosine antibodies 4G10, before stripping and reprobing with anti-STAT3 antibodies to confirm equal loading of STAT3. Multiple analyses of at least three independent clones of each mutant gave equivalent results.
