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).

从细胞中提取的30μg蛋白质样品经上样后，于8%十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）中完成分离，随后转印至硝酸纤维素膜上。将膜以5%脱脂牛奶进行封闭，再使用5%牛奶-PBS缓冲液稀释的抗SP1抗体（1:1000，货号9389，美国马萨诸塞州细胞信号技术公司（Cell Signaling Technology））或抗CCR7抗体（1:1000，货号ab32527，英国剑桥阿贝克隆比公司（Abcam））于4℃条件下孵育过夜；之后采用结合了DyLight荧光染料680的山羊抗兔二抗（货号A23720，中国武汉Abbkine公司）进行孵育。经PBST缓冲液充分洗涤后，借助Odyssey CLx红外成像系统（美国内布拉斯加州LI-COR公司）对蛋白条带进行可视化成像。