Li Jie, Zhang Jianchao, Wang Yanzhuang
Department of Molecular, Cellular and Developmental Biology, University of Michigan, 1105 North University Avenue, Ann Arbor, MI 48109, USA.
Department of Molecular, Cellular and Developmental Biology, University of Michigan, 1105 North University Avenue, Ann Arbor, MI 48109, USA; Department of Neurology, University of Michigan School of Medicine, Ann Arbor, MI, USA.
STAR Protoc. 2023 May 5;4(2):102283. doi: 10.1016/j.xpro.2023.102283.
N-Glycosylation is a common protein modification catalyzed by a series of glycosylation enzymes in the endoplasmic reticulum and Golgi apparatus. Here, based on a previously established Golgi α-mannosidase-I-deficient cell line, we present a protocol to investigate the enzymatic activity of exogenously expressed Golgi α-mannosidase IA in interphase and mitotic cells. We describe steps for cell surface lectin staining and subsequent live cell imaging. We also detail PNGase F and Endo H cleavage assays to analyze protein glycosylation. For complete details on the use and execution of this protocol, please refer to Huang et al..
N-糖基化是一种常见的蛋白质修饰，由内质网和高尔基体中的一系列糖基化酶催化。在此，基于先前建立的高尔基体α-甘露糖苷酶-I缺陷细胞系，我们提出了一种方案，用于研究间期和有丝分裂细胞中外源表达的高尔基体α-甘露糖苷酶IA的酶活性。我们描述了细胞表面凝集素染色及随后的活细胞成像步骤。我们还详细介绍了PNGase F和内切糖苷酶H切割试验以分析蛋白质糖基化。有关本方案使用和执行的完整详细信息，请参阅Huang等人的文章。
