Marks David L, Singh Raman Deep, Choudhury Amit, Wheatley Christine L, Pagano Richard E
Department of Biochemistry and Molecular Biology, Mayo Clinic and Foundation, Rochester, MN 55905, USA.
Methods. 2005 Jun;36(2):186-95. doi: 10.1016/j.ymeth.2004.12.001.
Sphingolipids (SLs) are concentrated at the plasma membrane where they play important roles in cell-cell communication, host-pathogen interactions, and cell signaling events. Our laboratory has used fluorescent SL analogs and SL-binding toxins to elucidate mechanisms by which SLs are internalized by endocytosis and subsequently sorted and transported to various intracellular compartments. These studies have relied on the use of temperature shift protocols, co-localization studies with compartment-specific markers, selective biochemical treatments that inhibit specific endocytic mechanisms, and the expression of dominant negative proteins (e.g., rabs) that block specific steps in transport. These methods are presented here so that they can be utilized by others for the study of endocytic trafficking of lipids and other molecules.
鞘脂（SLs）集中于质膜，在细胞间通讯、宿主-病原体相互作用及细胞信号事件中发挥重要作用。我们实验室已使用荧光鞘脂类似物和鞘脂结合毒素来阐明鞘脂通过内吞作用内化，随后被分选并转运至各种细胞内区室的机制。这些研究依赖于温度转换方案的使用、与区室特异性标记物的共定位研究、抑制特定内吞机制的选择性生化处理，以及阻断运输中特定步骤的显性负性蛋白（如Rabs）的表达。此处介绍这些方法，以便其他人可将其用于研究脂质和其他分子的内吞运输。
