Anti tyrp 2

Arbutin, 3-isobutyl-1-methylxanthine (IBMX), L-DOPA (L-3,4-dihydroxyphenylalanine), sodium hydroxide, thiazolyl blue tetrazolium bromide (MTT), O-tetradecanoyl phorbol-13-acetate (TPA), 2,2’-azobis(2-amidinopropane) dihydrochloride (AAPH), 8-methoxypsoralen (8-MOP), 3-Isobutyl-1-methyl-2,6(1H,3H)-purinedione (IBMX), and hydroquinone were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). All other reagents and chemicals were high-grade and commercially available. Antibodies bought from Bioworld Technology (St. Louis Park, MN, USA) included anti-Tyrosinase, anti-TYRP-1, anti-TYRP-2, anti-MITF, anti-phospho-p38, and anti-p38, anti-phospho-ERK, and anti-ERK, anti-phospho-JNK, and anti-JNK.

Melan-a cell lysates were prepared using a standard protocol, mixed with 5X SDS-PAGE (3M Science, Seoul, Korea) sample buffer and denatured at 100 °C for 5 min. An equal amount (20 µg protein) of each sample was separated by 10% SDS-PAGE gel electrophoresis, followed by electrotransfer to nitrocellulose membranes (Whatman, Dassel, Germany). The membranes were then incubated overnight with 5% skim milk (for anti-Tyr, anti-TYRP-1, anti-TYRP-2, anti-MITF) or 5% bovine serum albumin (BSA) for anti-phospho-JNK, anti-JNK, anti-phospho-p38, anti-p38, and Anti-p44/42MAPK (ERK1/2), and anti-phospho-p44/42MAPK (ERK1/2), detection. anti-Tyr, anti-TYRP-1, anti-TYRP-2, anti-MITF (Bioworld Technology, St. Louis Park, MN, USA) for Western blotting, and anti-phospho-JNK, anti-JNK, anti-phospho-p38, anti-p38, and Anti-p44/42MAPK (ERK1/2), and anti-phospho-p44/42MAPK (ERK1/2), and anti-MITF (Cell Signaling Technology, Beverly, MA, USA) for signaling studies were utilized as 1st antibodies. Anti-goat IgG-horse radish peroxidase (HRP) and anti-mouse IgG-HRP were purchased from Santa Cruz and used as 2nd antibodies. The reaction was proceeded using an ECL system (Perkin Elmer).