Anti tyrosinase

Penicillin/streptomycin (P/S), trypsin–ethylenediamine tetra-acetic acid (EDTA), and FBS were purchased from Gibco BRL (Gaithersburg, MD, USA) and Dulbecco’s modified eagle medium (DMEM) and phosphate-buffered saline (PBS) from Welgene (Daegu, Korea). Triton X-100, L-DOPA, bovine serum albumin (BSA), α-MSH, phenylmethylsulfonyl fluoride, NaOH, 10× DMEM, Tween-20 and dimethyl sulfoxide (DMSO) were from MilliporeSigma (St. Louis, MO, USA). Recombinant human epidermal growth factor (EGF: purity > 97%) was obtained from R&D Systems (Minneapolis, MN, USA). EZ-CyTox kits were supplied by DoGenBio (Seoul, Korea) and type I collagen by BD Bioscience (Franklin Lakes, NJ, USA). The antibodies used were as follows: anti-ERK1/2, anti-phospho ERK1/2, anti-AKT, anti-phospho AKT, anti-JNK, anti-phospho JNK, anti-p38 MAPK, anti-phospho p38 MAPK, anti-rabbit IgG, anti-MITF, and anti-mouse IgG (all from Cell Signaling, Beverly, MA, USA); polyclonal anti-type I, and IV collagen, monoclonal anti-type I and IV collagen (Abcam; Cambridge, UK); β-actin (MilliporeSigma); and anti-tyrosinase (Santa Cruz Biotechnology, CA, USA).

Hispolon was purchased from Enzo Life Sciences, Inc. (Farmingdale, NY, USA). Camptothecin, vitamin C, mushroom tyrosinase, L-DOPA, α-MSH, dimethyl sulfoxide (DMSO), sodium hydroxide (NaOH), sodium dodecyl sulfate (SDS) and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), α-modified essential medium (α-MEM), fetal bovine serum (FBS), penicillin, streptomycin and trypsin-EDTA were purchased from Gibco BRL/Invitrogen (Carlsbad, CA, USA). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was purchased from Affymetrix/USB (Cleveland, OH, USA). The β-arbutin was purchased from Alfa Aesar (Ward Hill, MA, USA). The anti-tyrosinase, anti-MITF and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The FITC-labeled annexin-V/PI apoptosis detection kit was purchased from BD Biosciences (San Jose, CA, USA). Caspases colorimetric assay kit was purchased from BioVision, Inc. (Milpitas, CA, USA). Deionized distilled water (ddH₂O) for solutions and buffers was obtained from the Milli-Q system (Millipore, Bedford, MA, USA).

Once the cells were harvested, the cell pellets were lysed in radioimmunoprecipitation (RIPA) buffer containing 1% NP-40, 1% sodium deoxycholate, and protease inhibitor (PI) cocktail on ice for 30 min. This was followed by centrifugation at 13,000 rpm for 30 min at 4 °C. The resulting supernatants were subsequently collected. Proteins were separated using 8% to 15% SDS polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred onto a polyvinylidene difluoride (PVDF) membrane. The membranes were blocked with 5% skim milk for 1 h at room temperature, followed by incubation with a primary antibody. Anti-Tyrosinase, anti-TRP1, anti-TRP2, anti-MITF, anti-phospho AKT, anti-AKT, anti-phospho ERK, anti-ERK, anti-phospho CREB, anti-CREB, and anti-actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Membranes were washed with Tris-buffered saline containing 0.1% Tween-20 (TBST) and incubated with donkey anti-rabbit or anti-mouse horseradish peroxidase (HRP)-conjugated IgG secondary antibody for 2 h. Protein bands were detected by an ImageQuant LAS 4000 mini (Fujifilm, Tokyo, Japan) and visualized using an image analysis program (Multi Gauge Ver. 3.0, Fujifilm, Tokyo, Japan).