Tyrosinase

Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin and streptomycin were purchased from Gibco BRL (Carlsbad, CA, USA). OEA, kojic acid, MK886, L-DOPA, dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), α-melanocyte-stimulating hormone (α-MSH), PD98059 and PTU were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). Antibodies against MC1R (1:1000, Cell Signaling Technology, Boston, USA), MITF (1:1000, Cell Signaling Technology), TRP-1 (1:1000, Cell Signaling Technology), Tyrosinase (1:1000, Cell Signaling Technology), PPARα (1:1000, Abcam, Cambridge, UK), total-ERK (1:1000, Cell Signaling Technology), phospho-ERK (Thr202/Thr204) (1:1000, Cell Signaling Technology), total-Akt (1:1000, Cell Signaling Technology), phospho-Akt (Ser473) (1:1000, Cell Signaling Technology), total-CREB (1:1000, Cell Signaling Technology), phospho-CREB (Ser133) (1:1000, Cell Signaling Technology), total-p38 (1:1000, Cell Signaling Technology), phospho-p38 (1:1000, Cell Signaling Technology) and mouse monoclonal anti-β-actin (1:10000, Sigma) were obtained from the indicated manufacturers. OEA, kojic acid, MK886, PD98059 and PTU were dissolved in dimethyl sulfoxide (DMSO), and the final DMSO concentration was less than 0.05%. L-DOPA was dissolved in phosphate buffered saline (PBS).

Protein samples isolated from B16F10 cells (30 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using 10-12% acrylamide gels and transferred to polyvinylidene fluoride membranes, which were then immediately placed in blocking buffer (5% non-fat milk) in 10 mM Tris (pH 7.5), 100 mM NaCl, and 0.1% Tween 20. The membrane was washed in TBS-Tween buffer for 30 min and then incubated with specific primary antibodies (1: 1000 dilution) indicated in the figure legends at 4°C overnight. After washing with TBS-Tween buffer, the membrane was incubated with a horseradish peroxidase-conjugated anti-mouse antibody (Santa Cruz, 1 : 10,000), an anti-rabbit antibody (Santa Cruz, 1 : 10,000), or an anti-goat antibody (Santa Cruz, 1 : 10,000) at 25°C for 1 h. The immunoblots were visualized using Western Bright Peroxide solution (Advansta, CA, USA) and ChemiDoc Touch imaging system (Bio-Rad, U.S.A) according to the manufacturer's instructions. Antibodies used in this study are as following: p-CREB (Santa Cruz-81486), CREB (Santa Cruz-81486), tyrosinase (Cell Signaling-104976), pMITF (Abcam-59201), MITF (Abcam-20663), p-p38 (Cell Signaling-921L), p38 (Cell Signaling-9212S), pERK (Cell Signaling-4370L), ERK (Cell Signaling-9201L), pJNK (Cell Signaling-9251L), JNK (Cell Signaling-9252S), and β-actin (Santa Cruz-47778).