2.8. Melanin measurement

Melanocytes transfected with SYT4‐CDS and Sh‐SYT4 were harvested and rinsed with PBS. For the assay of the total alkali‐soluble melanin (ASM), 1 mL of 0.2M NaOH was added, and the absorbance of the solution was measured spectrophotometrically at 475 nm. For the assay of eumelanin (EM), the cells were hydrolysed in hot 30% hypophosphoric acid and hydriodic acid. After cooling, 50% ethanol was added, and the cell samples were centrifuged at 2, 234 g for 10 minutes. Insoluble eumelanic pigments were selectively solubilized in hot sodium hydroxide and hydrogen peroxide, and the solution was cleared by centrifuging it at 10 , 700 g for 1 minute. The absorbance of the supernatant was measured at 350 nm. For pheomelanin (PM) assay, the cells were solubilized in phosphate buffer (pH 10.5) and cleared by centrifugation at 10, 700 g for 10 minutes before the determination of the absorbance at 400 nm. The melanin levels were normalized to the total number of cells. All experiments were performed in triplicate.