Fig. 2. Effects of overexpression and deletion of APC on ERK activity. (A) Left, DLD-1 cells were transfected with 0.5 µg of pCMV or with 0.25 or 0.5 µg of pCMV-APC. Cells were harvested 72 hours after transfection, and p-ERK, APC, ERKs and -tubulin were detected in whole-cell lysates by western blotting. The right bar graph shows quantitative analyses of the intensities of p-ERKs shown as the average of three independent identical experimental results for transfection with 0.5 µg of pCMV-APC from the left panel. (B) Apc^flox/flox primary mouse embryonic fibroblast (MEF) cells were infected with RCAS-Cre retrovirus (see Materials and Methods). ß-catenin, p-ERK, p-Raf1, p-Akt and -tubulin were detected in whole-cell lysates by western blotting. The right panels shows the Apc deletion within the Cre virus-infected MEF cells identified by RT-PCR analysis. Control is the result of PCR in the absence of template.