Fig. 5. Inhibition of nuclear export leads to a decrease in v-Src-mediated STAT3 activation and gene induction. (A) MEF^/STAT3-YFP cells were stimulated with 20 ng/ml IL-6 and 0.5 µg/ml sIL-6R for the indicated times and treated with 100 ng/ml ratjadone A (lower panel) or left untreated (upper panel). Fixed cells were analyzed by confocal laser-scanning microscopy. Bars, 10 µm. A western blot of corresponding lysates of MEF^/STAT3-YFP cells was analyzed for pY⁷⁰⁵-STAT3 and counterstained for total STAT3. (B) MEF^/STAT3-YFP cells were transfected with v-Src and treated for 2 hours with ratjadone A as indicated. Whole cell lysates were subjected to SDS-PAGE and western blotting. STAT3 tyrosine phosphorylation was detected with a pY⁷⁰⁵-STAT3 antibody. For loading control the blot was stained with a STAT3 antibody. (C) MEF^/ cells and MEF^/STAT3-YFP cells were transfected with v-Src and treated with ratjadone A for 24 hours. Cell lysates were subjected to SDS-PAGE and western blotting. pY⁷⁰⁵-STAT3, STAT3 and cyclin D1 were detected by immunostaining. (D) A luciferase reporter construct under control of the cyclin D1 promoter was transfected into MEF^/ and MEF^/STAT3-YFP cells. v-Src was co-transfected as indicated. 48 hours after transfection cells were treated with 100 ng/ml ratjadone A for 24 hours or left untreated. Reporter gene assays were performed in triplicate and s.d. was calculated.