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Fig. 6. The effects of ERK pathway inhibitors on Wnt3a-induced Raf-1, MEK, and ERK kinase activation. (A) NIH3T3 cells were grown in DMEM and either not treated or treated with 150 ng/ml recombinant Wnt3a (left panel) or with 100 µl Con-CM or Wnt3a-CM (right panel) for 30 minutes before harvesting. Where required, 20 µM U0126 was added 1 hour before the Con-CM or Wnt3a-CM treatment. (B) NIH3T3 cells were grown in DMEM and transfected with 0.5 µg pCMV vector, 0.5 µg pSVSport1-dn-Raf-1, 0.5 µg pCMV-MEK2A vector, 0.5 µg pMTSM-MycMKP-3 (left panel) and 0.5 µg pcDNA3.0 vector or pcDNA3.1 H-Ras N17 (right panel). 48 hours after transfection, the cells were treated for 30 minutes with 100 µl Con-CM or Wnt3a-CM. Cells were harvested and subjected to western blot analysis.
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