Lack of specificity of commercial antibodies leads to misidentification of angiotensin type 1 receptor protein.
Lack of specificity of commercial antibodies leads to misidentification of angiotensin type 1 receptor protein.
复制标题
DOI：
10.1161/hypertensionaha.112.203679
复制
发表时间：
2013-01
期刊：
影响因子：
--
通讯作者：
Coffman TM
中科院分区：
文献类型：
--
作者：
Herrera M;Sparks MA;Alfonso-Pecchio AR;Harrison-Bernard LM;Coffman TM
血管紧张素II型1受体（AT1R）介导血管紧张素II的大多数高血压作用。为了了解AT1受体在正常生理和病理生理中的分子调控，需要对AT1R蛋白进行灵敏、特异的检测。在这里，我们检查了一组假定的抗at1r抗体的特异性，这些抗体通常被该领域的研究人员使用。在这些研究中，我们对缺乏主要小鼠AT1R亚型AT1A （AT1AKO）或同时缺乏AT1A和AT1B亚型（AT1ABKO）的WT小鼠和转基因小鼠的肾组织进行了Western blotting和免疫细胞化学。对于测试的3种抗体，WT肾脏蛋白匀浆的Western blots产生了不同的条带，其大小范围为AT1R。此外，这些条带在缺乏一种或两种小鼠AT1R异构体的小鼠样本中出现相同。此外，WT小鼠的肾脏、肝脏和肾上腺的免疫组化染色模式与完全缺乏所有AT1受体的AT1ABKO小鼠非常相似。我们通过以下方法验证了每个小鼠系中AT1R亚型的缺失：1)定量PCR记录了mRNA种类的缺失；2)功能上通过评估血管紧张素ii依赖性血管收缩，这在AT1AKOs和AT1ABKOs中都被大大减弱。最后，这些抗体未能检测到HEK细胞中表位标记的AT1AR蛋白过表达。我们得出结论，商业来源的抗at1r抗体和已发表的研究中常用的抗at1r抗体在小鼠组织中表现出非特异性结合，这可能导致错误的结果。
The angiotensin II type 1 receptor (AT1R) mediates most hypertensive actions of angiotensin II. In order to understand the molecular regulation of the AT1 receptor in normal physiology and pathophysiology, methods for sensitive and specific detection of AT1R protein are required. Here, we examined the specificity of a panel of putative anti-AT1R antibodies that are commonly used by investigators in the field. For these studies, we carried out Western blotting and immunocytochemistry with kidney tissue from WT mice and genetically modified mice lacking the major murine AT1R isoform, AT1A (AT1AKO), or with combined deficiency of both the AT1A and AT1B isoforms (AT1ABKO). For the 3 antibodies tested, Western blots of protein homogenates from WT kidneys yielded distinct bands with the expected size range for AT1R. In addition, these bands appeared identical in samples from mice lacking one or both murine AT1R isoforms. Additionally, the pattern of immune histo-chemical staining in kidneys, liver and adrenal glands of WT mice was very similar to that of AT1ABKO mice completely lacking all AT1 receptors. We verified the absence of AT1R subtypes in each mouse line by: 1) quantitative PCR documenting the absence of mRNA species and, 2) functionally by assessing angiotensin II-dependent vasoconstriction, which was substantially blunted in both AT1AKOs and AT1ABKOs. Finally, these antibodies failed to detect epitope-tagged AT1AR protein over-expressed in HEK cells. We conclude that anti-AT1R antibodies available from commercial sources and commonly used in published studies exhibit non-specific binding in mouse tissue that may lead to erroneous results.