Figure 3

Activation of a conditionally active form of A-Raf(ΔA- Raf:ER) induces proliferation of human HaCaT keratinocytes via activation of the ERK signaling pathway. (A, B) Stimulation of HaCaT-ΔA- Raf:ER cells with either EGF (1 ng/ml) (A) or 4OHT (25 nM) (B) leads to an upregulation of DNA synthesis, as measured by the incorporation of the pyrimidine analogue 5-bromo-2'-deoxyuridine (BrdU) instead of thymidine into the DNA of proliferating cells. The incorporated BrdU was detected by immunoassay. Mitogenic signaling induced by EGF or 4OHT was completely abrogated by the MAP kinase kinase inhibitor PD98059. (C) Kinetics of ERK activation in EGF and 4OHT treated HaCaT-ΔA-Raf:ER cells. Whole cell extracts were prepared from cells at different time points and subjected to Western blot analysis. The blots were incubated with an affinity purified rabbit antibody directed against the phosphorylated (active) form of ERK2 (reproduced from [19] with copyright permission from the American Physiological Society).