Fig. 8. Overexpression of serine mutant STAT3 using recombinant adenovirus. (A) Keratinocytes were infected for 1 hour using recombinant adenoviruses encoding ß-Gal and STAT3 serine mutant (SmS). After 18 hours, total cell lysates were subjected to western blot analysis utilizing anti-STAT3 (top panel), anti-STAT3-p-tyr (middle panel) and STAT3-p-ser (bottom panel) antibodies. (B,C) Keratinocytes were infected for 1 hour using recombinant adenoviruses encoding WT STAT3, DN STAT3 and STAT3 serine mutant (SmS). After 24 hours, cells were (B) left untreated or (C) stimulated with insulin for the designated time periods (0, 5, 15 or 30 minutes). HA immunoprecipitates were subjected to western blot analysis and probed with anti-STAT3-p-tyr (top panel), anti-STAT3-p-Ser (middle panel) and anti-STAT3 (bottom panel) antibodies. Relative optical density of three representative blots is presented in arbitrary units (mean ± s.d.). (D) Keratinocytes were infected for 1 hour using recombinant adenoviruses encoding WT STAT3 and STAT3 serine mutant (SmS). Cells were stimulated with insulin for 5 and 30 minutes. Lysates were immunoprecipitated with PKC antibody and analyzed by western blotting, using anti-HA. Equal loading of gels was confirmed by reblotting with PKC antibody. Relative optical density of blots is presented in arbitrary units (mean ± s.d.). Experiments were repeated at least three times.