Antibody against mitf

Roswell Park Memorial Institute 1640 (RPMI-1640) medium, fetal bovine serum (FBS), penicillin and streptomycin were obtained from Life Technologies, Grand Island, NY, USA. Mushroom tyrosinase (EC 1.14.18.1), α-MSH, _L-DOPA, dimethyl sulfoxide (DMSO), and 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St Louis, CA, USA). Forskolin was obtained from Selleckchem (Houston, TX, USA). Antibodies against TYR, TRP-1, TRP-2, β-actin, and GAPDH were purchased from Santa-Cruz Biotechnology, Dallas, TX, USA. Antibody against MITF was obtained from Abcam, Cambridge, UK. Horseradish peroxidase (HRP)-linked anti-mouse IgG and anti-rabbit IgG anti-bodies were obtained from Cell Signaling Technology, Danvers, MA, USA. All other chemicals were reagent grade or HPLC grade and supplied by either Merck (Darmstadt, Germany) or Sigma-Aldrich.

Cells were harvested and lysed in radioimmunoprecipitation assay (RIPA) buffer (1% NP-40, 150 mM sodium chloride [NaCl], 10 mM Tris-HCl [pH 8.0], and 1 mM ethylenediaminetetraacetic acid [EDTA]) with a complete protease inhibitor (Sigma). The proteins were separated with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the gel transferred onto a polyvinylidene fluoride membrane (Millipore). Antibody against MITF was purchased from Abcam (Cambridge, UK), while those against tyrosinase, β-actin, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were obtained from Santa Cruz Biotechnology (Dallas, TX). Antibodies against p-Erk1/2 and Erk1/2 were provided by Cell Signalling Technology (Danvers, MA).