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Figure 2 | Cell Communication and Signaling

Figure 2

From: Dominant-negative activity of the STAT3-Y705F mutant depends on the N-terminal domain

Figure 2

Influence of STAT3-YF-YFP on the phosphorylation of endogenous STAT3 and analysis of binding to a gp130 phosphopeptide. (A) Inducible HEK cells stably transfected with STAT3-YF-YFP were incubated with increasing concentrations of doxycycline (Dox) for 5 h. The cells were stimulated with 20 ng/ml IL-6 and 1 μg/ml sIL-6Rα for the indicated times or left unstimulated. Cellular lysates were analyzed by immunoblotting using antibodies against pY705-STAT3, STAT3, pERK1/2, ERK1/2 and GAPDH as a loading control. Signal intensities of STAT3, STAT3-YF-YFP and pY705 STAT3 were quantified. (B) HEKgp80 cells were transfected with STAT3-YFP, STAT3-YF-YFP or STAT3-R609Q-YFP. The cells were stimulated with 20 ng/ml IL-6 for 30 minutes or left unstimulated. The lysates were incubated with a biotinylated gp130-receptor-peptide containing phosphotyrosine 767 coupled to avidin-Sepharose. The precipitates were analyzed by immunoblotting using a specific antibody against STAT3. The expression of the transfected STAT3 constructs was verified by immunoblotting of untreated lysates.

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