Tyrosinase sc 20035

Proteins were obtained using RIPA buffer containing protease inhibitors and then quantified. The proteins (40 µg) were electrophoresed by 8% SDS-PAGE gels and transferred to nitrocellulose. The membranes were blocked with 5% skim milk in PBST (PBS containing 0.1% Tween 20) at RT for 1 h, washed with PBST, incubated with primary antibodies (1:1000) for 16 h at 4 °C, washed with PBST, incubated with HRP-conjugated secondary antibodies for 1.5 h at RT. Western blotting was performed using the following antibodies; MITF antibody(sc-56725, Santa Cruz Biotechnology, Santa Cruz, CA, USA), TRP-1(sc-166857, Santa Cruz Biotechnology), tyrosinase (sc-20035, Santa Cruz Biotechnology), p-ERK (4370, Cell Signaling Technology, Beverly, MA, USA), p-GSK3β (5558, Cell Signaling Technology), β-Catenin (9562, Cell Signaling Technology), and GAPDH (5174, Cell Signaling Technology). Anti-mouse and anti-rabbit IgG antibodies were from Santa Cruz Biotechnology. The protein-antibody complex was visualized by an ECL system. Bands were quantified using the FluorChem E system image analyzer (Cell Biosciences, Santa Clara, CA, USA). GAPDH was used as an internal control. For quantify, images of protein bands were measured using Image J software (NIH, Bethesda, MD, USA).