Anti trp 2

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).

Cells were fixed with 4% paraformaldehyde for 20 min at room temperature followed by 0.1% Triton X-100 to allow cell permeabilization. Cells were then incubated with the following primary antibodies: anti-PPARγ, anti-SREBP1, anti-RUNX2, anti-Mitf, anti-Tyrosinase, anti-TRP2 (Santa Cruz Biotechnology, USA), anti-Sox2, anti-Nestin antibody (Merck Millipore, Germany), for 1 hour. Primary antibodies were visualized using anti-rabbit IgG, anti-goat or anti-mouse IgG Alexa Fluor 488 (BD Bioscences). Nuclei were visualized with DAPI. Fluorescence signals were recorded using a CCD camera (Zeiss, Oberkochen, Germany).