Fig. 7. Insulin-induced STAT3 nuclear translocation is abrogated by PKC inhibition. Primary keratinocytes were plated on glass slides and maintained for 5 days in low-Ca²⁺ MEM (0.05 mM) until they reached 80% confluence. (A) Cells were left untreated (upper panel) or pre-treated with 5 µM rottlerin (Rott) for 7 minutes (lower panel), followed by 10^–7 M insulin for 5 and 30 minutes (Ins). Cells were fixed in methanol, washed and air-dried. Cultures were analyzed by immunofluorescence using anti-phospho-tyr-705-STAT3 (STAT3-p-tyr) antibody, followed by FITC-conjugated secondary antibody. Slides were viewed by confocal microscopy. (B) Cells were untreated (–) or treated with insulin (Ins) for 5 and 30 minutes. Following treatment, cells were fixed in methanol and analyzed by immunofluorescence using anti-PKC antibody followed by FITC conjugated secondary antibody and scanned by confocal microscope. Arrow in middle panel indicates translocation of PKC to the perinuclear membranes. Experiments were repeated at least three times. Bar, 20 µm.