The effects of upstream signaling pathways on ERK1/2 activation in Grey horse melanoma cells. (A) Levels of NRAS, BRAF, MEK1/2, SPROUTY2 and RKIP expression in Grey horse vs. human melanoma cell lines analyzed by Western blot. (B) The effects of PI3 kinase/AKT and RAF pathways on ERK1/2 activation were analyzed using inhibitors LY294002 (50 μM) and L779450 (10 μM), respectively. The cell lines were cultured 19 in the presence of DMSO as vehicle control or with the indicated concentrations of the inhibitors for 12 h following a 2 h serum-free preincubation and their effect on ERK1/2 activation was analyzed by Western blot. Similar results were obtained in three independent experiments. (C) The effect of the RAF kinase inhibitor L779450 (10μΜ) on cell growth of GHM cell lines was measured in relation to DMSO-treated control by Alamar Blue assay 72 h post-treatment. The mean ± s.e. of three independent experiments performed in triplicates is presented. ***P <0.001. (D) The efficiency of L779450 (10μΜ) treatment on cell growth was determined by calculating the concentration necessary to achieve a 50% reduction in cell proliferation (IC50). The data represent the mean ± s.e. of three independent experiments performed in triplicates.