Ab 3312

The expressions of melanogenic proteins were determined using the western blotting analysis which was modified from a previous study (16 (link)). Briefly, 40 μig of protein lysate obtained from treated B16 cells was electrophoresed and transferred to polyvinylidene difluoride membrane (Millipore, Germany). The membrane was treated individually with primary antibody including rabbit polyclonal anti-MITF, 1:1000 (ab 20663, Abcam, UK); rabbit polyclonal anti- TYR, 1:1000 (ab 180753, Abcam, UK); mouse monoclonal anti-TRP-1, 1:100 (ab 3312, Abcam, UK); rabbit polyclonal anti-TRP-2, 1:100 (ab 74073, Abcam, UK), and rabbit polyclonal anti-β-actin, 1:1,000 (ab 8227, Abcam, UK) at room temperature for 1 h with agitation before washing with 1× tris-buffered saline and Tween® 20 (TBST), pH 7.2 for three times. The membrane was then treated with secondary antibody including polyclonal goat anti-rabbit IgG-horseradish peroxidase(HRP), 1:1,000 (P0448, Dako, Denmark) and polyclonal goat anti-mouse IgG-HRP, 1:1,000 (P0447, Dako, Denmark) at room temperature for 1 h with gentle agitation. Then, washed the m emb rane with 1× TBST, pH 7.2 for 5 min three times, and incubated with Luminata Forte western HRP substrate (Merck, Germany) for 10 min. The proteins were then visualized using a gel documentation analyzer (ImageQuant LAS 500, UK) and analyzed the densitometry by Image J software.