Activation of the ERK pathway in melanoma of Grey horses. (A) Double immunofluorescence staining was performed for P-ERK1/2 (green in a1-a2, c1-c2) or total ERK1/2 (green in b1-b2, d1-d2) and a nuclear melanocytic marker, MITF (red in a2, b2, c2, d2) in melanoma tissue sections from non-grey and Grey horses; a3, b3, c3, d3, corresponding bright field images of the sections. Arrows and arrowheads indicate representative cells for the corresponding stains. Scale bar, 10 μm. (B) Quantification of P-ERK1/2 and ERK1/2 immunofluorescent signals in relation to the total number of MITF+ cells in melanoma tissue sections from non-grey (n = 12; dark grey bars) and Grey (n = 19; light grey bars) horses shown as the mean ± s.e. of three independent experiments. ***P <0.001. (C) Western blot analysis of P-ERK1/2 levels in GHM vs. human melanoma cell lines with Q61RRAS (BL), V600EBRAF (Mel-Ho) and WTRAS WTBRAF (M5). (D) Quantification of total ERK1/2-normalized P-ERK1/2 protein levels in the cell lines listed in (C). The mean ± s.e. of three independent Western blots are shown.