Trp 2

Dimethyl sulfoxide (DMSO), α-MSH, NaOH, MTT, radioimmunoprecipitation assay (RIPA) buffer and l-DOPA were obtained from Sigma–Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin, trypsin–ethylenediaminetetraacetic acid and PD98059 were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Antibodies against tyrosinase, TRP-1, TRP-2 and MITF were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). LY294002 and antibodies against p-p38, p38, p-JNK, JNK, p-ERK, ERK, p-AKT, AKT and β-actin were obtained from Cell Signaling Technology (Danvers, MA, USA). An enhanced chemiluminescence (ECL) kit and 2× Laemmli sample buffer were obtained from Biosesang (Sungnam, Gyeonggi-do, Korea) and Bio-Rad (Hercules, CA, USA), respectively.

Primary antibodies for mouse anti-MITF (#sc-25386), tyrosinase (#sc-15341), TRP-1 (#sc-514900), TRP-2 (#sc-74439), p-ERK (#sc-7383), ERK 1/2 (#sc-514302), and β-actin (#sc-47778) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The primary anti-bodies for mouse anti-cyclic AMP (cAMP) response element-binding protein (CREB) (8763) (#9198S) and CREB (D76D11) (#4820S) were purchased from Cell Signaling Technology (Beverly, MA, USA); all primary antibodies were used at 1:1000 dilutions. Secondary mouse antibody for primary antibodies was purchased from Enzo Life Sciences (Farmingdale, NY, USA) and used at 1:5000 dilutions. Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), and penicillin/streptomycin were purchased from Gibco BRL (Carlsbad, CA, USA). Most chemicals, including α-MSH, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), L-DOPA, 1,1-diphenyl-2-picryl-hydrazy (DPPH), 2, 2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS), arbutin, and mushroom tyrosinase were purchased from Sigma-Aldrich (St. Louis, MO, USA).

The cells were washed with PBS and centrifuged at 1000× g for 5 min at 4 °C. The obtained pellets were resuspended in RIPA buffer with protease and proteasome inhibitors, incubated on ice for 20 min, sonicated, and centrifuged at 20,000× g for 20 min at 4 °C. The supernatants were separated using 8% or 10% SDS-PAGE and then transferred to nitrocellulose membranes. The membranes were incubated overnight at 4 °C with primary antibodies against MITF, GAPDH (Thermo Fisher Scientific, Rockford, IL, USA), tyrosinase, TRP1, TRP2, β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), cAMP-responsive element binding protein (CREB), or pCREB (Cell Signaling Technology, Danvers, MA, USA), which was followed by incubation with the appropriate secondary antibodies (Thermo Fisher Scientific, Rockford, IL, USA). The blots were developed by using the EZ-Western Lumi Femto™ western blot detection kit (Daeil Lab Service, Seoul, Korea).

Sigma-Aldrich (St. Louis, MO, USA) provided the 6-methylcoumarin (CAS 92-48-8), 7-methylcoumarin (CAS 2445-83-2), 4-hydroxy-6-methylcoumarin (CAS 13252-83-0), 4-hydroxy-7-methylcoumarin (CAS 18692-77-8), α-melanocyte-stimulating hormone (α-MSH), a protease/phosphatase inhibitor cocktail, sodium hydroxide (NaOH), and L-DOPA. Biosesang (Seongnam, Gyeonggi-do, Korea) supplied the MTT, DMSO, PBS, TBS, SDS, a RIPA buffer, and an ECL kit. Thermo Fisher Scientific (Waltham, MA, USA) provided the DMEM, penicillin–streptomycin, a BCA protein assay kit, and 0.5% trypsin-ethylenediaminetetraacetic acid (10×). The primary antibodies, tyrosinase, TRP-1, TRP-2, and MITF, were purchased from Santa Cruz Biotechnology (Dallas, TX, USA), and the other antibodies, p-ERK, ERK, p-p38, p38, p-JNK, JNK, p-PKA, PKA, p-AKT, AKT, p-GSK-3β, GSK-3β, p-β-catenin, β-catenin, β-actin, and antirabbit, and secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Skim milk was purchased from BD Difco (Sparks, MD, USA), the fetal bovine serum (FBS) was purchased from Merck Millipore (Burling, VT, USA), and the Tween 20 and 2× Laemmle sample buffer were obtained from Bio-rad (Hercules, CA, USA).