Department of Medical Microbiology and Immunology, School of Medicine, University of California, Davis, Davis, CA, USA.
Laboratory of Viral Diseases, NIAID NIH, Bethesda, MD, USA.
Methods Mol Biol. 2025;2854:265-282. doi: 10.1007/978-1-0716-4108-8_25.
Protein kinase R (PKR), a key double-stranded RNA (dsRNA)-activated sensor, is pivotal for cellular responses to diverse stimuli. This protocol delineates a comprehensive methodological framework employing single luciferase assays, yeast assays, immunoblot assays, and quantitative PCR (qPCR) to discern and validate PKR activities and their downstream impacts on NF-κB-activating signaling pathways. These methodologies furnish a systematic approach to unraveling the role of PKR as a dsRNA sensor and effector in antiviral innate immunity, enabling in-depth analyses of dsRNA sensor activities.
蛋白激酶 R（PKR）是一种关键的双链 RNA（dsRNA）激活传感器，对于细胞对各种刺激的反应至关重要。本方案描述了一种综合的方法学框架，包括使用单个荧光素酶测定、酵母测定、免疫印迹测定和定量 PCR（qPCR）来辨别和验证 PKR 活性及其对 NF-κB 激活信号通路的下游影响。这些方法提供了一种系统的方法来揭示 PKR 作为 dsRNA 传感器和抗病毒先天免疫效应子的作用，能够深入分析 dsRNA 传感器的活性。
