Xia Mingfeng, Edwards Tessa, Rong Shunxing
Center for Human Nutrition, University of Texas Southwestern Medical Center, Dallas, TX, USA.
Department of Endocrinology and Metabolism, Zhongshan Hospital, Fudan University, Shanghai, China.
Bio Protoc. 2024 Dec 20;14(24):e5139. doi: 10.21769/BioProtoc.5139.
Sterol regulatory element binding proteins (SREBPs) are transcription factors that reside in the endoplasmic reticulum (ER) membrane as inactive precursors. To be active, SREBPs are translocated to the Golgi where the transcriptionally active N-terminus is cleaved and released to the nucleus to regulate gene expression. Nuclear SREBP levels can be determined by immunoblot analysis; however, this method can only determine the steady-state levels of nuclear SREBPs and does not capture the actual status of activation. The vesicle budding assay provides an alternative way to quantify the activation of SREBPs by monitoring the initiation of SREBP translocation from the ER to the Golgi through vesicles. Microsomal membranes isolated from the liver are incubated in a reaction buffer containing the necessary components to facilitate vesicle formation. Microsomal membranes and vesicles are isolated and SREBPs are quantified in each by immunoblot analysis. The amount of SREBPs found in the budded vesicles provides an assessment of the SREBP activation in the liver. Key features • This protocol describes a method to isolate budding vesicles from liver ER membranes. • The in vitro budding assay can be applied to investigate the movement of proteins from the ER to the Golgi. • This protocol was developed based on the procedures described previously with cultured cells [1-3].
固醇调节元件结合蛋白（SREBPs）是以内质网（ER）膜上无活性前体形式存在的转录因子。为了激活，SREBPs会转运至高尔基体，在那里转录活性N端被切割并释放到细胞核以调节基因表达。核SREBP水平可通过免疫印迹分析来确定；然而，该方法只能确定核SREBPs的稳态水平，无法捕捉其实际激活状态。囊泡出芽试验提供了一种通过监测SREBP通过囊泡从内质网转运至高尔基体的起始过程来量化SREBPs激活的替代方法。从肝脏分离的微粒体膜在含有促进囊泡形成所需成分的反应缓冲液中孵育。分离微粒体膜和囊泡，并通过免疫印迹分析对每个样本中的SREBPs进行定量。在出芽囊泡中发现的SREBPs量可评估肝脏中SREBP的激活情况。关键特性 • 本方案描述了一种从肝脏内质网膜分离出芽囊泡的方法。 • 体外出芽试验可用于研究蛋白质从内质网到高尔基体的转运。 • 本方案是基于先前针对培养细胞所描述的程序开发的[1 - 3]。
