Kennedy Donna, Samali Afshin, Jäger Richard
Apoptosis Research Centre (ARC), National University of Ireland, Biosciences Research Building, Corrib Village, Dangan, Galway, Ireland.
Methods Mol Biol. 2015;1292:3-18. doi: 10.1007/978-1-4939-2522-3_1.
Many experimentally induced or disease-related cellular dysfunctions stress the endoplasmic reticulum, commonly resulting in an accumulation of unfolded proteins in the ER lumen which is sensed by three ER-resident transmembrane proteins, PERK, ATF6, and IRE1. Their activation by such ER stress affects the unfolded protein response, which consists of a shutoff of protein translation and at the same time the switching-on of specific transcription factors that control genes which function to reduce the burden of unfolded proteins to the ER. Here, we describe two sets of methods for monitoring the occurrence of ER stress and UPR signaling in human cells by analyzing markers of activation of all three ER stress sensor proteins. The first set of methods is based on the qualitative and quantitative analysis of UPR-induced transcripts by qPCR. The second set of methods consists of Western blot-based analysis of UPR-induced proteins or protein modifications. Their combined analysis allows assessment of activation of all three ER stress-activated signaling pathways that in combination are characteristic for the UPR.
许多实验诱导或疾病相关的细胞功能障碍会给内质网带来压力，通常会导致内质网腔中未折叠蛋白的积累，这会被三种内质网驻留跨膜蛋白PERK、ATF6和IRE1感知。它们被这种内质网应激激活后会影响未折叠蛋白反应，该反应包括蛋白质翻译的关闭，同时开启特定转录因子，这些转录因子控制着一些基因，这些基因发挥作用以减轻内质网中未折叠蛋白的负担。在这里，我们描述了两组通过分析所有三种内质网应激传感器蛋白激活标记来监测人类细胞内质网应激和未折叠蛋白反应（UPR）信号发生情况的方法。第一组方法基于通过qPCR对未折叠蛋白反应诱导转录本进行定性和定量分析。第二组方法包括基于蛋白质印迹法对未折叠蛋白反应诱导蛋白或蛋白质修饰进行分析。它们的联合分析可以评估所有三种内质网应激激活信号通路的激活情况，这些信号通路共同构成了未折叠蛋白反应的特征。
