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Melanocytes were transfected twice at days 1 and 3 for a total of 5 days with the indicated siRNAs. Cells were then collected, sonicated in 50 mM Tris-HCl pH 7.4, 2 mM EDTA, 150 mM NaCl, 1 mM dithiothreitol (with the addition of protease inhibitor cocktail, Roche) and pelleted at 20,000×g for 15 min at 4 °C. The pigment was rinsed once in ethanol:ether (1:1) and dissolved in 2 M NaOH with 20% (v/v) DMSO at 60 °C. Melanin content was measured by optical density at 490 nm (Spectramax 250, Molecular Devices).
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