Fig. 1. Insulin induces STAT3 activation and nuclear translocation in mouse primary keratinocytes. (A) Keratinocytes following 5 days in culture were stimulated with 10^–7 M insulin for the times indicated. STAT3 immunoprecipitates were probed with anti-p-tyr-STAT3 (top panel). Equal loading of gels was confirmed by reblotting with STAT3 antibody (bottom panel). Relative optical density of four representative blots is presented in arbitrary units (mean ± s.d.). (B) Keratinocytes were plated on glass slides. Cultures (5 days old) were stimulated with insulin for 5 minutes, fixed in ethanol and analyzed by immunofluorescence, using anti-p-tyr-STAT3, followed by FITC-conjugated secondary antibody. Cells were viewed by confocal microscopy. (C) Keratinocytes were stimulated with 10^–7 M insulin for the times indicated. STAT3 immunoprecipitates were probed with anti-IRß antibody (top panel) and with anti-IGF 1Rß antibody (middle panel). Equal loading of gels was confirmed by reblotting with STAT3 antibody (bottom panel). The experiment was repeated twice. Bar, 20 µm.