Western blot

Proteins (30μg) extracted from cells were loaded and separated in 8% SDS-PAGE and then transferred on nitrocellulose membranes. The membranes were blocked with 5% non-fat milk and incubated with Anti-SP1 (1:1000, 9389, Cell Signaling Technology, MA, USA) or Anti-CCR7 (1:1000, ab32527, Abcam, Cambridge, UK) in 5% milk/PBS buffer at 4℃ overnight, followed by incubation with goat anti-rabbit secondary antibody (A23720, Abbkine, Wuhan, China) conjugated with DyLight fluorescent 680. After extensive wash with PBST buffer, the band of protein was visualized under Odyssey CLx imaging system (LI-COR, Inc., NE, USA).