Fig. 5. Overexpression of WT STAT3 and DN STAT3 using recombinant adenoviruses. (A,B,C) Primary keratinocytes were infected for 1 hour using ß-galactosidase (ß-Gal), WT STAT3 and DN STAT3 recombinant adenoviruses containing a hemagglutinin (HA) tag, and experiments were conducted 24 hours after infection. (A) Total cell lysates were subjected to western blot analysis using anti-STAT3 antibody. (B) Overexpressing cells were either untreated (–) or treated with insulin for 5, 15 and 30 minutes. Lysates were immunoprecipitated with anti-HA antibody and immunoprecipitates analyzed by western blotting, using a anti-p-tyr-STAT3 antibody. (C) Overexpressing cells were treated with insulin for 5 and 30 minutes. PKC immunoprecipitates were analyzed by western blotting, using anti-HA and anti-STAT3 antibodies. Equal loading of gels was confirmed by reblotting with PKC antibody. Relative optical density of three representative blots is presented in arbitrary units (mean ± s.d.). (D) Keratinocytes were either infected (OE) for 1 hour with WT STAT3 adenovirus constructs or double infected with DN PKC followed by WT STAT3 recombinant adenovirus infection. 24 hours following infection, cells were untreated (–) or treated with insulin for 5 minutes. HA immunoprecipitates were subjected to western blot analysis and probed with anti-phosphotyrosine and anti-HA antibodies. Experiments were repeated at least three times.