Anti tyrosinase

Cell lysates were mixed with SDS buffer (3 M, Maplewood, Minnesota, USA), and denatured at 100 °C for 5 min using a standard protocol. Adequate amount of proteins (30 µg) were separated using 10% SDS-polyacrylamide gel electrophoresis and blotted onto nitrocellulose membranes (Whatman, Dassel, Germany). The membranes were blocked with 5% non-fat skim milk in TBS-T buffer, followed by incubation with primary antibodies in 5% skim milk. Primary antibodies, such as anti-MITF, anti-CREB, anti-tyrosinase, anti-TRP-2, anti-p-CREB, anti-TRP-1, and anti-β-actin were purchased from Bioworld Technology (St. Louis Park, MN, USA). Anti-mouse IgG-horseradish peroxidase (HRP) and anti-goat IgG-HRP from Santa Cruz Biotechnology was used as secondary antibody. An ECL solution system (Perkin Elmer) was used to detect the antigen-antibody reaction.

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 mixed with sample buffer (250 mM Tris-HCl (pH 6.8), 0.5 M DTT, 10% SDS, 0.5% bromophenol blue, 50% glycerol, 5% 2-mercaptoethanol), and denatured at 100 °C for 5 min using a standard protocol. A 10% of SDS-PAGE was used to separate the sample proteins (50 µg). Following electrotransfer to nitrocellulose membranes (Whatman, Dassel, Germany), the membranes were immersed overnight in a mixture containing antibodies and 5% skim milk. Primary antibodies, such as anti-tyrosinase, anti-TRP-1, anti-TRP2, anti-MITF, p-CREB, and total CREB and β-actin, were from Bioworld Technology (St. Louis Park, MN, USA). Secondary antibodies (anti-goat IgG-horse radish peroxidase (HRP) and anti-mouse IgG-HRP) were purchased from Santa Cruz. The resulting reaction was exposed using an ECL solution system (Perkin Elmer).