title: Analysis of SCAP N-glycosylation and Trafficking in Human Cells.
abstract: Elevated lipogenesis is a common characteristic of cancer and metabolic diseases. Sterol regulatory element-binding proteins (SREBPs), a family of membrane-bound transcription factors controlling the expression of genes important for the synthesis of cholesterol, fatty acids and phospholipids, are frequently upregulated in these diseases. In the process of SREBP nuclear translocation, SREBP-cleavage activating protein (SCAP) plays a central role in the trafficking of SREBP from the endoplasmic reticulum (ER) to the Golgi and in subsequent proteolysis activation. Recently, we uncovered that glucose-mediated N-glycosylation of SCAP is a prerequisite condition for the exit of SCAP/SREBP from the ER and movement to the Golgi. N-glycosylation stabilizes SCAP and directs SCAP/SREBP trafficking. Here, we describe a protocol for the isolation of membrane fractions in human cells and for the preparation of the samples for the detection of SCAP N-glycosylation and total protein by using western blot. We further provide a method to monitor SCAP trafficking by using confocal microscopy. This protocol is appropriate for the investigation of SCAP N-glycosylation and trafficking in mammalian cells.
authors.affiliation: Department of Radiation Oncology, The Ohio State University Comprehensive Cancer Center and College of Medicine.
authors.forename: Chunming
authors.initials: C
authors.lastname: Cheng
authors.affiliation: Department of Radiation Oncology, The Ohio State University Comprehensive Cancer Center and College of Medicine.
authors.forename: Jeffrey Yunhua
authors.initials: JY
authors.lastname: Guo
authors.affiliation: Department of Radiation Oncology, The Ohio State University Comprehensive Cancer Center and College of Medicine.
authors.forename: Feng
authors.initials: F
authors.lastname: Geng
authors.affiliation: Department of Radiation Oncology, The Ohio State University Comprehensive Cancer Center and College of Medicine.
authors.forename: Xiaoning
authors.initials: X
authors.lastname: Wu
authors.affiliation: Department of Radiation Oncology, The Ohio State University Comprehensive Cancer Center and College of Medicine.
authors.forename: Xiang
authors.initials: X
authors.lastname: Cheng
authors.affiliation: Department of Radiation Oncology, The Ohio State University Comprehensive Cancer Center and College of Medicine.
authors.forename: Qiyue
authors.initials: Q
authors.lastname: Li
authors.affiliation: Department of Radiation Oncology, The Ohio State University Comprehensive Cancer Center and College of Medicine; Deliang.Guo@osumc.edu.
authors.forename: Deliang
authors.initials: D
authors.lastname: Guo
mesh_terms: D018929:Cell Culture Techniques; D002462:Cell Membrane; D002784:Cholesterol; D004268:DNA-Binding Proteins; D004721:Endoplasmic Reticulum; D006031:Glycosylation; D006056:Golgi Apparatus; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D021381:Protein Transport; D014157:Transcription Factors
chemical_list: D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; C102196:SREBP cleavage-activating protein; D014157:Transcription Factors; D002784:Cholesterol