Western blot analysis
The protein samples were prepared from B16 cells as described above and separated via 10% SDS-PAGE. The gels were transferred to PVDF membranes, blocked with 5% skim milk or 5% BSA, and incubated overnight at 4 °C with appropriate antibodies. Following incubation with the secondary antibody, the protein bands were detected using an enhanced chemiluminescence (ECL) western blotting detection kit, and the signals were quantified using a ChemiDoc MP Imaging system (Bio-Rad Laboratories Inc. Hercules, CA, USA). All experiments were performed five times.