Different kinetics of Stat4 activation in response to IL-12 and IFN- in Th1 cells. Th1 and Th2 cells were cultured in a medium supplemented with IL-2 and stimulated with IL-12 or IFN- and cells were harvested, washed, and lysed as described in Materials and Methods. Five micrograms of whole cell extracts were incubated with a³²P-labeled oligonucleotide with the sequence of a Stat4 DNA-binding site. Migration of specific Stat4: DNA complexes is indicated by the arrow. (A) Kinetics of inducible Stat4 DNA-binding activity upon stimulation of Th1 and Th2 cells with either IL-12 or IFN- for 0.25, 1, 4, and 16 hours. (B) Inducible Stat4 DNA-binding activity in Th1 (lanes 1-3) and Th2 cells (lanes 4-6) before (lanes 1 and 4) or 30 minutes after stimulation with IL-12 (lanes 2 and 5) or IFN- (lanes 3 and 6). Nuclear extracts from IL-12–stimulated Th1 cells were incubated with anti-Stat4 (lane 7) or control (lane 8) rabbit polyclonal antibodies. (C) A densitometric analysis of the kinetics of Stat4 DNA-binding activity induced by IL-12 or IFN- in Th1 cells from the experiment shown in (A).