Total protein extraction reagent

Total protein from mice skin tissues or melanocytes was extracted using total protein extraction reagent (Beyotime, Shanghai, China) according to the manufacturer’s instructions, and the concentration was measured using a nucleic acid/protein analyzer. Two hundred micrograms of protein lysate from each sample were size-separated by SDS-PAGE electrophoresis and transferred onto nitrocellulose filter membranes (Boster, Wuhan, China). The membranes were blocked in 5% skimmed milk powder (Boster, Wuhan, China) at room temperature for 1 h and then were probed with the primary antibody diluted in Tris-buffered saline-Tween (TBST) overnight at 4 °C. The next day, the membranes were washed 3 times in TBST for 10 min each and incubated with HRP-conjugated second antibody (1:10,000 (v/v), Boster, Wuhan, China) at 37 °C with horizontal shaking for 1 h. Subsequently, the membranes were washed 6 times in TBST for 5 min each, and the proteins were visualized via a super ECL chemiluminescence solution (Boster, Wuhan, China). The Western blot results were analyzed using Image-ProPlus 6.0 software (Olympus, Hatayaga, Japan) to measure the area and gray value for each target band. The target protein expression level was normalized relative to the corresponding internal reference level in each lane.