FIGURE 5.

MAP kinase activation occurs after expression of Smad dominant-negative expression vector. Inhibition of Smad3 activation was achieved by transient transfection with a c-myc-tagged dominant-negative Smad3 expression vector. A: Transfection efficiency of the dominant-negative Smad3 expression vector. Histogram of FL-1 fluorescence in EGFP-transfected versus mock-transfected cells. Transfection efficiency, based on the proportion of cells in gated region M1, is estimated at 55%. Efficacy of the expression vector was confirmed by co-transfection of 1.0 μg of the dominant-negative (DN) expression vector or empty vector (EV) together with 0.9 μg of the Smad-responsive (SBE)₄-Lux reporter and 0.1 μg of the Renilla luciferase construct using 6 μl of the mixed lipofection reagent FuGene6. B: Twenty-four hours after transfection cells were stimulated with 1 ng/ml TGF-β1 for 6 hours before quantitation of luciferase content. Results are expressed as ratios of firefly/Renilla luciferase and represent mean ± SD, n = 3. In parallel experiments, cells were transiently transfected with 1 μg of the c-myc-tagged dominant-negative Smad3 expression vector using 3 μl of FuGene6. Twenty-four hours after transfection, cells were stimulated with TGF-β1 (1 ng/ml) for 30 minutes. Subsequently, total cell extracts were generated, and immunoblot analysis of lysate samples for ERK/total-ERK (C) and phosphorylated p38/total p38 (D) was performed. E: In parallel experiments, overexpression of the vector was confirmed by c-myc immunoblot.