@inbook{f74a153c868d40a292eda7be17d27cfe,
title = "Assaying Sterol-Regulated ER-to-Golgi Transport of SREBP Cleavage-Activating Protein Using Immunofluorescence Microscopy",
abstract = "Sterol regulatory element-binding proteins (SREBPs) are a family of membrane-bound transcription factors that regulate the uptake and synthesis of cholesterol and fatty acids in mammalian cells. SREBP cleavage-activating protein (SCAP) is an endoplasmic reticulum (ER) protein that binds newly synthesized SREBP, retaining it in the ER where SREBP is inactive. SCAP binds cholesterol and functions as the cholesterol sensor in this regulatory system. Under low cholesterol conditions, SCAP escorts SREBP from the ER to the Golgi apparatus where two proteases sequentially cleave and activate SREBP. Given their central importance in maintaining cellular lipid homeostasis, other mechanisms exist to regulate SREBP activity, such as control of protein synthesis and degradation. Here, we describe methods to assay ER-to-Golgi transport of SCAP in vitro using immunofluorescence microscopy and two different cell systems, Chinese hamster ovary (CHO) cells stably expressing hamster GFP-SCAP and human HeLa cells transiently expressing human GFP-SCAP. These methods will permit investigators to determine if cellular perturbations act by affecting the ER-to-Golgi transport of SCAP.",
keywords = "Cholesterol, ER-to-Golgi transport, Immunofluorescence, Lipid homeostasis, SREBP cleavage-activating protein (SCAP), Sterol regulatory element-binding protein (SREBP)",
author = "Ishida, {Chiaki T.} and Wei Shao and Espenshade, {Peter J.}",
note = "Funding Information: Financial Support This work was supported by the National Institutes of Health (HL077588 and GM126088, PJE). Publisher Copyright: {\textcopyright} 2023, The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.",
year = "2023",
doi = "10.1007/978-1-0716-2639-9_45",
language = "English (US)",
series = "Methods in Molecular Biology",
publisher = "Humana Press Inc.",
pages = "755--764",
booktitle = "Methods in Molecular Biology",
}