IFNs induce Stat1 phosphorylation, attenuate Stat3 phosphorylation, and repress IL-6-induced DNA binding of Stat3 in favor of Stat1. (A-B) U-266-1970 cells were either treated with IL-6 (20 U/mL) alone or in combination with IFN-γ (1000 U/mL; A) or IFN-α (1000 U/mL; B). At the indicated time points, the cells were harvested and protein extracts were prepared and analyzed by Western blot using antibodies specific for Tyr701 phosphorylated Stat1, Tyr705 phosphorylated Stat3, Stat1, Stat3, and β-actin. (C) U-266-1970 cells were treated with IL-6 (20 U/mL) alone or in combination with IFN-γ (1000 U/mL) or IFN-α (1000 U/mL) for the indicated time points. Nuclear extracts were prepared and DNA-binding complexes were analyzed by EMSA, as described in “Patients, materials, and methods,” using a 32P-labeled m67SIE probe, containing a GAS site. The positions of Stat3/Stat3, Stat1/Stat3 and Stat1/Stat1 DNA-binding complexes are indicated by the supershift (Stat3) and reduction of the complexes (Stat1) as a result of the addition of specific antibodies (lanes 10 and 11). The experiments in 7A-C were performed 3 times.