Fig. 5. Pharmacological inhibition of conventional and novel PKC isoforms blocks HGF/SF-induced PKC phosphorylation but fails to inhibit HGF/SF-mediated VEGF/VPF gene expression. (A) Detection of phosphorylated PKC- (p-PKC-, upper panel) or the respective actin protein expression (from Santa Cruz) by western blot analysis. HaCaT cells were left untreated or were exposed to HGF/SF (for 10 minutes at 100 ng/ml) after preincubation with broad-range PKC inhibitor calphostin C (Cal. C at 1 µM for 60 minutes), isotype-selective (conventional and novel) PKC inhibitor bisindolylmaleimide I (Bis. I at 1 µM for 60 minutes) or solvent only (DMSO, 0.1%) as indicated. Experiments were repeated twice with similar results. (B) Analysis of CAT expression derived from a transiently transfected -88/+54 bp VEGF/VPF-promoter-based construct. HaCaT cells were cultured in the absence or presence of bisindolylmaleimide I (Bis. I at 1 µM, starting 1 hour prior to HGF/SF treatment) without growth factor stimulation or with HGF/SF treatment (for 16 hours at 100 ng/ml) as indicated. Data displayed represent the means±s.d. of three independent triplicate assays. (C) VEGF/VPF protein of supernatants derived from confluent HaCaT cells. Cells were cultured in the absence or presence of calphostin C (at 1 µM, starting 1 hour prior to HGF/SF treatment) without growth factor stimulation or with HGF/SF treatment (for 24 hours at 100 ng/ml) as indicated. Data from three independent triplicate experiments are expressed as ng secreted VEGF/VPF protein per mg total cellular protein (mean±s.e.m.).