Fig. 6. Targeting PKC- expression attenuates HGF/SF-induced VEGF/VPF promoter activity. (A) Western blot analyses of HaCaT cells that were left untreated (media change) or were stimulated by HGF/SF (10 minutes at 100 ng/ml). Cellular extracts were immunoprecipitated (IP) by a specific anti-PKC- antibody (Santa Cruz) prior to immunoblotting (IB). Immunoblotting with pan-PKC- antibody (Santa Cruz; upper panel) and with phospho-PKC- antibody (lower panel). (B) Analysis of firefly luciferase (Luc) expression derived from a transiently transfected -88/+54 bp VEGF/VPF-promoter-based reporter construct (1 µg) along with antisense oligonucleotides directed against the translation start site of PKC- (second bar) or of PKC- (third bar, each at 0.1 µM). Twenty-four hours after transfection, HaCaT cells were stimulated with HGF/SF (at 100 ng/ml) for 16 hours or were left untreated. This assay is representative of three independent sets of experiments revealing comparable results. Fold increase in HGF/SF-induced luciferase activity is calculated on the basis of data obtained from the respective controls, which were left unstimulated. Values represent the mean±s.d. of triplicate assays. Statistical analyses were performed on data from three experiments (Student's t-test, ^*P<0.05). (C) Analysis of firefly luciferase expression derived from a transiently transfected -88/+54 bp VEGF/VPF-promoter-based reporter construct (2.5 µg) along with wild-type PKC- (wt-PKC-) or its kinase-deficient mutant (mut-PKC-; 500 ng each) vector. Twenty-four hours after transfection, HaCaT cells were stimulated with HGF/SF (at 100 ng/ml) for 16 hours or were left untreated. Data displayed herein include the values of three independent duplicate experiments (mean±s.e.m.; Student's t-test, ^*P<0.05).

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