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.