Fig. 1. v-Src expression leads to constitutive activation of wild-type and fluorescent STAT3 fusion proteins. (A) COS-7 cells were transiently transfected with v-Src and STAT3 as indicated and stimulated with 20 ng/ml IL-6 and 1 µg/ml sIL-6R for 30 minutes or left untreated. Whole cell lysates were subjected to SDS-PAGE and western blotting. STAT3 tyrosine phosphorylation was detected with a pY⁷⁰⁵-STAT3 antibody and as a loading control STAT3 was counterstained with a specific STAT3 antibody. (B) Transiently expressed STAT3-YFP and mutant fusion proteins as indicated were precipitated from COS-7 cell lysates with a GFP antibody that recognizes YFP and CFP. Western blot analyses of precipitates were performed for pY⁷⁰⁵-STAT3 and counterstained for total STAT3 and YFP. hc, heavy chain of the precipitating antibody. (C) Nuclear extracts were prepared from MEF^fl/fl, MEF^/ or MEF^/STAT3-YFP cells stimulated with 20 ng/ml IL-6 and 1 µg/ml sIL-6R for 30 minutes as indicated. STAT3, STAT1 and STAT3-YFP DNA-binding to a [³²P]-labelled SIE-probe was detected by EMSA. Antibodies against STAT3 and GFP were used for the supershift experiment. (D) MEF^/STAT3-YFP cells were co-transfected with v-Src, fixed and stained for pY⁴¹⁶-Src. Fixed cells were analyzed by confocal laser-scanning microscopy. Bars, 10 µm. A Western blot of lysates of MEF^/STAT3-YFP cells was analyzed for pY⁷⁰⁵-STAT3 and counter-stained for total STAT3. (E) MEF^/STAT3-CY cells were treated and analyzed as described in D. Bars, 10 µm. A Western blot of lysates of MEF^/STAT3-CY cells was analyzed for pY⁷⁰⁵-STAT3 and counter-stained for total STAT3. (F) MEF^fl/fl and MEF^/STAT3-YFP cells were transfected with v-Src, Fyn, Abl, or BCR-Abl or left untransfected for IL-6 stimulation and the negative control as indicated. Whole cell lysates were subjected to western blotting and analyzed for pY⁷⁰⁵-STAT3 and total STAT3 as a loading control.