Anti mitf primary antibody

The cell lysates were separated on 10% gels by SDS-PAGE electrophoresis and then transferred to nitrocellulose filter membranes (Millipore, New York, NY, USA). After the membranes were blocked in 5% evaporated skimmed milk (Boster, Wuhan, China) at 25 °C for 1 h and washed five times using Tris-buffered saline-Tween (TBST) for 8 min each time, it was incubated with anti-Sox5 primary antibody at 1:800 dilution (from rabbit, Abcam, Cambridge, MA, USA), anti-MITF primary antibody at 1:500 dilution (from rabbit, Abcam, Cambridge, MA, USA), anti-TYR primary antibody at 1:1000 dilution (from rabbit, Abcam, Cambridge, MA, USA) and anti-β-actin primary antibody at 1:2000 dilution (from rabbit, Boster, Wuhan, China) overnight at 4 °C, respectively. Following washing four times in TBST for 8 min each, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit-IgG at 1:2000 (Boster, Wuhan, China) at 37 °C for 1.5 h. Subsequently, a super ECL chemiluminescence plus (Boster, Wuhan, China) was used for visualization after the membranes were washed. The relative intensities of the above proteins were analyzed by Image Lab software (Bio-Rad Laboratories, Philadelphia, PA, USA).