Fig. 1. Biochemical and functional characterization of STAT3-YFP. (A) Domain structures of STAT3 and STAT3-YFP. Domain borders were chosen according to Becker et al. (Becker et al., 1998). (B) The fusion protein STAT3-YFP is a functional player. COS-7 cells were cotransfected with expression vectors encoding STAT3 or STAT3-YFP and chimeric IL-5R/gp130 receptor chains as indicated. Cells were stimulated with IL-5 (20 ng/ml) for 30 minutes or left unstimulated. Lysates were analyzed by western blotting (WB) using a STAT3 phosphotyrosine-specific antibody (upper panel). After stripping, the blot was reprobed with a STAT3 antibody (lower panel). (C) Cells transfected as described above were stimulated with IL-5 (20 ng/ml) for various time periods as indicated. STAT3 DNA-binding activity was monitored by an EMSA using the sis-inducible element (SIE)-probe. Supershifts were performed using STAT3 (^*) or GFP (#) antibodies. (D) HepG2 cells were cotransfected with a STAT3-specific luciferase-reporter gene plasmid (m67-SIE-TK-luc), a plasmid encoding ß-gal for determination of transfection efficiency, and mock vector, STAT3, or STAT3-YFP as indicated. Cells were stimulated for 16 hours with 20 ng/ml IL-6 (dark gray bars) or left untreated (light gray bars). Relative luciferase activities were normalized with ß-gal activities. Mean values of experiments performed in triplicate are depicted.