Subcellular Fractionation of Melanocytes

Subcellular fractionation was carried out using a protocol described earlier (Mahanty et al., 2016 (link)). Briefly, melanocytes were harvested, washed with 1XPBS and suspended in 0.25 M sucrose buffer (0.25 M sucrose, 1 mM EDTA, 25 mM HEPES pH 7.4, 0.02% sodium azide and protease inhibitor cocktail). Cells were homogenized on ice using Dounce homogenizer and then clarified by centrifugation at 600 g for 10 min at 4°C. The cell lysate was fractionated on a sucrose step gradient (2.0 M, 1.6 M, 1.4 M and 1.2 M sucrose buffers manually layered from bottom to top in ultracentrifuge tube) using SW55TI rotor by spinning at 160000 g at 4°C for 4 - 6 h in a Beckman L-80 ultracentrifuge. Fractions were manually separated and subjected to immunoblotting. In Fig. S4, percent enrichment of each protein in the fraction was calculated from the protein band densities, normalized with 1^st fraction and then plotted as a graph.