Figure 3
From: Dominant-negative activity of the STAT3-Y705F mutant depends on the N-terminal domain
Dimer-formation of STAT3-YF-YFP. (A) Inducible HEK cells stably transfected with STAT3-YF-YFP were incubated with 10 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 30 minutes or left unstimulated. The lysates were incubated with a specific antibody against an epitope comprising phosphotyrosine 705 of STAT3 coupled to Protein-G-Sepharose as indicated. Precipitates were analyzed by immunoblotting using a STAT3 antibody. Expression of the STAT3 constructs in the whole cell lysates (WCL) was verified by immunoblotting. (B) HEKgp80 cells were transfected with different YFP-labelled STAT3 constructs. The cells were stimulated with 20 ng/ml IL-6 for 30 minutes or left unstimulated. The lysates were incubated with Coomassie brilliant blue G-250 and separated on a 4-16% gradient gel under non-denaturing conditions. The gel was analyzed on a fluorescence scanner. Homo- as well as heterodimers are marked with arrows. Asterisks highlight STAT3-YF-YFP homodimers and WT-STAT3/STAT3-YF-YFP heterodimers as well as the corresponding homo- and heterodimers containing STAT3-ΔN-YFP.