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Fig. 2. STAT3 in nuclear bodies. (A) Untransfected HepG2 cells (upper panels) or HepG2 cells transfected with STAT3 (lower panels) were stimulated with 20 ng/ml IL-6 for 15 minutes or left unstimulated as indicated. Cells were fixed and STAT3 was immunostained with a STAT3-specific antibody and a TRITC-conjugated secondary antibody. Images were taken by confocal microscopy. Endogenous (upper panels) as well as transfected (lower panels) STAT3 was detected. Bars, 10 µm. (B) STAT3-YFP nuclear translocation analyzed by live cell imaging. HepG2 cells transfected with STAT3-YFP were placed in a perfusion chamber at 37°C and analyzed by confocal laser scanning microscopy. Cells were stimulated with 20 ng/ml IL-6 and pictures of a single cell were taken every 30 seconds. Images taken at the timepoints indicated are depicted. Bar, 10 µm. A time-lapse movie can be downloaded as supplementary data on the homepage of the Journal of Cell Science (jcs.biologists.org). (C) Kinetics of STAT3 and STAT3-YFP phosphorylation. HepG2 cells were transfected with mock vector or STAT3-YFP. Lysates were prepared after stimulation with 20 ng/ml IL-6 for the timepoints indicated. Endogeneous STAT3 (endo) and STAT3-YFP were precipitated by a STAT3-specific antibody (upper panels). STAT3-YFP was precipitated by a GFP-specific antibody (lower panels). Tyrosine phosphorylation of STAT3 and STAT3-YFP was detected by western blot analysis using a STAT3 phosphotyrosine-specific antibody. After stripping the blots were reprobed with a STAT3 antibody for loading control.
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