Protein was harvested from zebrafish melanomas 2–6 weeks post-tumor onset by first euthanizing the fish with tricaine methanesulfonate followed by surgical removal of the tumor. Tumors were triturated and lysed in ice-cold RIPA buffer containing a cOmplete protease inhibitor tablet (Roche). Protein concentration was measured using the Pierce BCA Protein Assay Kit (Life Technologies). Samples were run on 10% polyacrylamide gels, transferred, and developed using fluorophore-conjugated antibodies (LI-COR). Antibodies against the following proteins were used: Mitfa (28 (link)); RAF-1 (c-RAF), pMEK1/2 (S217/221), MEK1/2, p44/42 MAPK (ERK1/2), alpha-Tubulin, pAKT S473, AKT, pc-KIT, c-KIT (Cell Signaling 9422, 9154, 8727, 4695, 38735, 4060, 4685, 3391, 3308, respectively); pERK (Sigma m8159); BRAFV600E (Spring Bioscience E19290); total BRAF (Millipore 10146); IRDye 800CW Donkey anti-Rabbit and IRDye 680RD Goat anti-Mouse (LI-COR 926-32213 and 926-68070, respectively). All quantitative measurements were calculated based on measurements from a LI-COR Odyssey Imaging System and quantified with Image Studio Lite (v 5.0) software. For background subtraction, a median pixel intensity for regions 3 pixels above and below a selected band was calculated and subtracted from the mean pixel intensity of that band.