MARCH6，也称为 TEB4 或 RNF176，是一种 E3 连接酶，嵌入内质网 (ER) 膜中，它使许多底物蛋白泛素化，使它们发生蛋白酶体介导的降解。近年来，MARCH6 已被确定为多种代谢途径的关键调节因子，包括胆固醇和脂滴稳态、蛋白质质量控​​制、铁死亡和肿瘤发生。尽管它很重要，但目前还没有特异性抗体可以检测和监测培养细胞和动物中的 MARCH6 水平。在这里，我们通过生成一种单克隆抗体来解决这一缺陷，该抗体可以特异性检测昆虫、小鼠、仓鼠和人类来源的培养细胞以及小鼠组织中的 MARCH6，并且与其他蛋白质的交叉反应性最小。然后我们使用该抗体来评估 MARCH6 的两个特性。首先，用该抗体对小鼠组织进行分析表明，肝脏中 MARCH6 的含量最高。其次，使用该抗体对五种不同细胞系的分析表明，随着细胞胆固醇含量的变化，MARCH6 的内源水平没有变化。该试剂有望成为探究 MARCH6 额外信号作用的有用工具。版权所有 © 2024 作者。由爱思唯尔公司出版。保留所有权利。

MARCH6, also designated as TEB4 or RNF176, is an E3 ligase that is embedded in membranes of the endoplasmic reticulum (ER) where it ubiquitinates many substrate proteins to consign them to proteasome-mediated degradation. In recent years, MARCH6 has been identified as a key regulator of several metabolic pathways, including cholesterol and lipid droplet homeostasis, protein quality control, ferroptosis, and tumorigenesis. Despite its importance, there are currently no specific antibodies to detect and monitor MARCH6 levels in cultured cells and animals. Here, we address this deficiency by generating a monoclonal antibody that specifically detects MARCH6 in cultured cells of insect, mouse, hamster, and human origin, as well as in mouse tissues, with minimal cross-reactivity against other proteins. We then used this antibody to assess two properties of MARCH6. First, analysis of mouse tissues with this antibody revealed that the liver contained the highest levels of MARCH6. Second, analysis of five different cell lines with this antibody showed that endogenous levels of MARCH6 are unchanged as the cellular content of cholesterol is varied. This reagent promises to be a useful tool in interrogating additional signaling roles of MARCH6.Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.