Fig. 5. Effects of ß-catenin overexpression on activation of the RAF1MEKERK cascade, and effects of PD98059 and constitutively active MEK on ß-catenin-induced ERK activation. (A) DLD-1 cells were transfected with 1.0 µg of pcDNA3.0 or Flag-ß-catenin-pcDNA3.0. After 48 hours, they were harvested and p-ERK, p-MEK, p-Raf (Ser-338), Flag-ß-catenin and -tubulin were detected by western blotting. (B) DLD-1 cells were transfected with 1.0 µg of pcDNA3.0, Flag-ß-catenin-pcDNA3.0 or Flag-S33Y-ß-catenin-pcDNA3.0. Where required, cells were exposed to 20 µM PD98059 for 24 hours before harvesting (upper panel), or 0.2 µg of MEK-CA-pcDNA3.0 was co-transfected with the Flag-ß-catenin-pcDNA3.0 (lower panel). After 48 hours p-ERK, Flag-ß-catenin and ERK were detected by western blotting.