Fig. 1.

pS727-dependent STAT3 inactivation for both pY705 dephosphorylation and post-activation nuclear export. (A) Changes in the pY705 level over time. HepG2-SKO cells expressing WT STAT3 (SKO+STAT3 WT), STAT3 S727A (SKO+STAT3 S727A) or STAT3 S727D (SKO+STAT3 S727D) were stimulated with 20 ng ml⁻¹ IL-6 for the indicated times, and whole cell extracts (WCEs) were subjected to immunoblot analysis with antibodies to phospho-Tyr705, phospho-Ser727, STAT3 and β-actin. Experiments were repeated three times. (B) Cellular distribution of STAT3. Cells were subjected to immunohistochemistry using a monoclonal antibody to STAT3 (red) and to DNA staining with DAPI (blue) and observed by confocal microscopy. One representative picture of each condition from >25 pictures is shown. The average percentage of nuclear STAT3 for each condition is shown. The scale bar in the first picture shows 10 μm. (C) STAT3 nuclear distribution. The data of the nuclear distribution of STAT3 in the indicated condition (shown as a box plot) were analyzed with one-way ANOVA. Pairwise comparisons using t-tests with pooled standard deviation were used. P-value <0.001 or <0.01 is depicted as *** or **, respectively, with a line showing the compared data sets. P-value >0.05 is shown as ns. The experiments in (B) and (C) were performed at least three times with similar results. (D) Changes in the pY705 level of endogenous STAT3 over time. HepG2 cells were stimulated with IL-6 for the indicated times, and WCEs were subjected to immunoblot analysis as in (A). (E) Nuclear distribution of endogenous STAT3 in HepG2 cells stimulated with IL-6 for the indicated times. The experiments were repeated twice with similar results.