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Fig. 4. Activation of ERK, MEK and Raf-1 kinases by Wnt3a in NIH3T3 and L cells. (A) NIH3T3 cells were grown in DMEM and treated with 150 ng/ml recombinant Wnt3a. Cells were harvested at 0, 30 minutes, 12 hours and 24 hours. p-ERK, p-MEK, p-Raf (Ser338), -tubulin, ß-catenin and cyclin D1 proteins were detected by western blot analysis. The p-Raf-1 (Ser338) bands differentially modified were marked with arrows. (B) Left panels, NIH3T3 cells were grown in DMEM and treated with 100 µl Wnt3a-CM (upper panel) or Con-CM (lower panel). Cells were harvested at 0, 5 minutes, 15 minutes, 30 minutes, 1 hour, 2, 4, 8, 12 and 24 hours. The p-ERK, p-MEK, p-Raf (Ser338), ß-catenin and -tubulin proteins were detected by western blot analysis (see A) Right panels, NIH3T3 cells were grown in DMEM and treated with different amounts of Wnt3a-CM (0, 1, 10, 50, 100 or 200 µl; upper panel) or Con-CM (0, 10, 50, 100 and 200 µl; lower panel). Cells were harvested 30 minutes after either Con-CM or Wnt3a-CM treatment for western blot analysis (see A). (C) Upper panel, L cells were grown in DMEM and treated with 100 µl Wnt3a-CM. Cells were harvested at 0, 5 minutes, 30 minutes, 2 and 8 hours after Wnt3a-CM treatment. Lower panel, L cells were grown in DMEM and treated with different amounts of Con-CM (0, 10, 50 or 100 µl). Cells were harvested 30 minutes after Con-CM treatment and western blot analyses were then performed.

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