Fig. 4. Insulin-induced PKC-STAT3 association and activation depends on PKC activity. (A) Keratinocytes were infected (OE) for 1 hour with recombinant ß-Gal (control), wild-type (WT) PKC and dominant-negative PKC (DN PKC) adenovirus constructs. Following infection, cells were incubated for 18 hours, and left untreated (–) or stimulated with insulin for 5 and 30 minutes. STAT3 immunoprecipitates were subjected to western blot analysis and probed with anti-PKC and anti-STAT3 antibodies. (B) Keratinocytes were either infected for 1 hour with ß-Gal or WT STAT3 adenovirus constructs, or double infected with DN PKC followed by WT STAT3 recombinant adenovirus infection. Following infection, cells were incubated for 24 hours and overexpressing cells were untreated (–) or stimulated with insulin for 5 and 30 minutes. PKC immunoprecipitates were subjected to western blot analysis and probed with anti-phosphoserine-727-STAT3 (STAT3-p-ser), anti-PKC and anti-STAT3 antibodies. (C) Keratinocytes were either uninfected (–) or infected (OE) for 1 hour with recombinant dominant-negative PKC (DN PKC) adenovirus. After 24 hours, cells were left untreated (–) or treated with insulin for 5 minutes. PKC immunoprecipitates were subjected to western blot analysis and probed with anti-phosphotyrosine-705-STAT3 (STAT3-p-tyr) and anti-PKC antibodies. Relative optical density of three blots are presented in arbitrary units (mean ± s.d.). Experiments were repeated at least three times.