Properties of tyrosine-dependent and -independent pathways of STAT3 activation. (A) Sensitivity of STAT3 activation to the Jak2 inhibitor AG-490 from WT, mDA, and mO G-CSF-Rs. Cells were starved as described in the presence (+) or absence (−) of AG-490 before stimulation for 10 minutes with (+) or without (−) G-CSF. To aid the interpretation of the result, exposures were adjusted so that G-CSF–treated samples were approximately equal. (B) Kinetics of STAT3 activation in Ba/F3 cells expressing WT, mDA, and mO G-CSF-Rs. Serum- and growth factor–deprived cells were incubated with 100 ng/mL G-CSF for the times indicated. Nuclear extracts were prepared and incubated with ³²P-labeled double-stranded m67 oligonucleotide. (C) Transactivation of STAT3(m67)-luciferase reporter by parental Ba/F3 cells (par), or Ba/F3 cells expressing WT or mutant G-CSF-Rs. Luciferase activity was assayed after incubation of cells from the same transfection with either G-CSF or IL-3. Activity is expressed as fold induction by G-CSF compared with by IL-3 to standardize for transfection efficiency of the reporter construct. Data represent the mean of at least three independent analyses, with the standard error indicated.