Anti trp1

B16 melanoma cells were seeded at a density of 5 × 10⁵ cells per 100-mm petri dish and cultivated overnight. The medium was then replaced by medium without (control) or with α-MSH (0.2 µM) or ALE (30 µg/mL). After incubation of B16 cells for 48 h or 72 h, RIPA buffer, containing a protease inhibitor cocktail, was used to harvest the total protein. Extracted protein samples (10 µg) were separated via SDS-PAGE, and transferred onto PVDF membranes. These membranes were incubated with anti-TYR, anti-TRP1, anti-DCT, or anti-GAPDH primary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA); then detected by adding anti-mouse or anti-goat secondary antibodies (LI-COR Biosciences, Lincoln, NE, USA). Proteins were visualized using the LiCor Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, 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).

Melan-a cell lysates were prepared using RIPA buffer supplemented with 1% phenylmethylsulfonyl fluoride and 1X protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). Lysates (40 µg) were resolved by SDS-PAGE and transferred to a nitrocellulose membrane. Proteins were measured using antibody-based probes, including anti-tyrosinase, anti-TRP-1, anti-TRP-2, and anti-MITF (Santa Cruz biotechnology, Dallas, TX, USA). GADPH was used as a loading control antibody.