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. 2013 Nov 5;11:83. doi: 10.1186/1478-811X-11-83

Figure 4.

Figure 4

Importance of the N-terminal domain for dominant-negative activity of the STAT3-YF mutant. (A) Inducible HEK cells stably transfected with STAT3-YF-ΔN-YFP were incubated with 10 or 20 ng/ml doxycycline (Dox) for 5 h or left untreated. The cells were stimulated with 20 ng/ml IL-6 and 1 μg/ml sIL-6Rα for the indicated times or left unstimulated. HEKgp80 cells were stimulated with IL-6 for 30 minutes or left unstimulated. Cellular lysates were analyzed by immunoblotting using antibodies against pY705-STAT3, STAT3 and GAPDH as a loading control. (B) Nuclear translocation of STAT3-CFP in the presence of STAT3-YF-ΔN-YFP after IL-6 stimulation. HEKgp80 cells were cotransfected with STAT3-YF-ΔN-YFP and STAT3-CFP. The cells were stimulated with 20 ng/ml IL-6 for 30 minutes or left unstimulated. All cells were fixed and nuclear translocation was analyzed by confocal microscopy. Scale bars represent 10 μm. (C) Real-time PCR analysis to examine the influence of STAT3-YF-ΔN-YFP on the STAT3-induced SOCS3 mRNA expression after IL-6 stimulation. Inducible HEK cells stably transfected with STAT3-YF-ΔN-YFP were incubated with 20 ng/ml doxycycline for 5 h and stimulated with 20 ng/ml IL-6 and 1 μg/ml sIL-6Rα for 60 minutes or left unstimulated. Stably transfected HEK cells without doxycycline treatment served as a control. RNA was isolated and SOCS3 mRNA expression analyzed by qRT-PCR. The diagram represents the mean of three independent measurements with standard deviations.