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Fig. 8. Activation of the ERK pathway by ß-catenin overexpression, and effects of dominant-negative Tcf-4 on ß-catenin-induced ERK pathway activation. (A) NIH3T3 cells were grown in DMEM and transfected with either 0.5 µg pcDNA3.0 vector or 0.5 µg Flag-ß-catenin-pcDNA3.0. Cells were harvested 48 hours after transfection. (B) NIH3T3 cells were grown in DMEM and transfected with either 0.5 µg pcDNA3.0 vector or 0.5 µg Flag-ß-catenin-pcDNA3.0. Where required, cells were treated with 20 µM U0126 1 hour before harvesting. Anti-Flag antibody was used for detection of Flag-ß-catenin. (C) NIH3T3 cells were grown in DMEM and transfected with either 0.5 µg pcDNA3.0 vector or 0.5 µg Flag-ß-catenin-pcDNA3.0. Cells were co-transfected with 0.5 µg of vector or {Delta}N-Tcf-4E together with Flag-ß-catenin-pcDNA3.0. The cells were harvested after 48 hours and western blot analyses were performed with ß-catenin, Flag, ERK, p-ERK, p-MEK, Raf-1 or {alpha}-tubulin antibodies.