Western Blotting of MC1R Protein

Western blotting was performed by standard methods.^{49 (link)} Cells were lysed in NP40 solution supplemented with 1mM Na₃VO₄, 1mM PMSF containing the protease inhibitor cocktail. Protein concentrations of lysates were calculated by the BCA (Bicinchoninic Acid) assay. Protein (approximately 30 μg) was diluted in a sample buffer [2.5% SDS, 10% glycerol, 5% β-mercapto-ethanol, 50mM Tris (pH = 6.8) and 0.1% bromophenol blue] and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A monoclonal rabbit anti-human anti-MC1R (Abcam) was diluted at 1:2000. β-Actin (1:10000, cat. # A5441, SIGMA) was utilized as a control for normalization of protein gel loading. Detection of proteins was done with peroxidase-conjugated anti-mouse (cat. # 7076S, Cell Signaling, 1:5000) or anti-rabbit IgG secondary antibodies (cat. # 7074S, Cell Signaling, 1:2000) and ECL (PerkinElmer).