Fig. 8. Inhibition of MEK1/2 blocks HGF/SF-induced PKC phosphorylation of ERK1/2 and inhibits HGF/SF-mediated VEGF/VPF gene transcription and protein expression. (A) Detection of phosphorylated ERK1/2 (p-ERK1/2, upper panel) or total ERK1/2 protein (ERK1/2, lower panel) 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 the MEK1/2 inhibitor PD 98059 (at 50 µM for 60 minutes) or solvent only (DMSO, 0.1%) as indicated. Experiments were repeated three times with comparable 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 PD 98059 (at 50 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. The data displayed represent the mean±s.d. of three triplicate assays. (C) VEGF/VPF protein of supernatants derived from confluent HaCaT cells. Cells were cultured in the absence or presence of PD 98059 (at 50 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 triplicate experiments are expressed as ng secreted VEGF/VPF protein per mg total cellular protein (mean±s.e.m.). Statistical analyses were performed on data from three sets of experiments (Student's t-test, ^**P<0.01, ^*P<0.05).