通过代谢标记和脉冲追踪分析监测组织蛋白酶D向溶酶体的靶向运输。
Monitoring the Targeting of Cathepsin D to the Lysosome by Metabolic Labeling and Pulse-chase Analysis.
作者信息
Tavares Lucas A, daSilva Luis L P
机构信息
Department of Cell and Molecular Biology, Ribeirão Preto Medical School (FMRP), University of São Paulo (USP), Ribeirão Preto, SP, Brazil.
出版信息
Bio Protoc. 2017 Nov 5;7(21):e2598. doi: 10.21769/BioProtoc.2598.
Abstract
Mannose 6-phosphate receptors function can be studied in living cells by investigating alterations in processing and secretion of their ligand Cathepsin D. The assay described here is well established in the literature and comprises the metabolic labeling of newly synthesized proteins with [S] methionine-cysteine in HeLa cells to monitor Cathepsin D processing through secretory pathway and secretion using immunoprecipitation, SDS-PAGE and fluorography.
摘要
甘露糖6-磷酸受体的功能可以通过研究其配体组织蛋白酶D的加工和分泌变化在活细胞中进行研究。这里描述的检测方法在文献中已有充分记载，包括在HeLa细胞中用[S]甲硫氨酸-半胱氨酸对新合成的蛋白质进行代谢标记，以通过免疫沉淀、SDS-PAGE和荧光自显影监测组织蛋白酶D通过分泌途径的加工和分泌。
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