State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Faculty of Medicine and Life Sciences, Xiamen University, Xiamen, Fujian 361102 China.
State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Faculty of Medicine and Life Sciences, Xiamen University, Xiamen, Fujian 361102 China; Research Unit of Cellular Stress of CAMS, Xiang'an Hospital of Xiamen University, Cancer Research Center of Xiamen University, School of Medicine, Faculty of Medicine and Life Sciences, Xiamen University, Xiamen, Fujian 361102 China.
STAR Protoc. 2023 Dec 15;4(4):102581. doi: 10.1016/j.xpro.2023.102581. Epub 2023 Sep 21.
Inflammasomes are multimeric protein complexes that have crucial functions in innate immunity. Here, we present a protocol to reconstitute the PELO-driven assembly of NAIP5-NLRC4 inflammasome in vitro. We describe steps for expression and purification of recombinant PELO and flagellin, preparation of native cell lysate containing NAIP5-NLRC4, and in vitro assembly of NAIP5-NLRC4 inflammasome. We then detail analysis of NAIP5-NLRC4 inflammasome by blue native polyacrylamide gel electrophoresis and immunoblotting. This protocol can be adapted to monitor the oligomeric assembly of other inflammasome types. For complete details on the use and execution of this protocol, please refer to Wu et al. (2023)..
炎症小体是多聚体蛋白复合物，在先天免疫中具有关键功能。在这里，我们提供了一个方案，用于在体外重新组装由 PELO 驱动的 NAIP5-NLRC4 炎症小体的组装。我们描述了表达和纯化重组 PELO 和鞭毛蛋白、制备含有 NAIP5-NLRC4 的天然细胞裂解物以及体外组装 NAIP5-NLRC4 炎症小体的步骤。然后，我们详细介绍了通过蓝色非变性聚丙烯酰胺凝胶电泳和免疫印迹分析 NAIP5-NLRC4 炎症小体。该方案可用于监测其他炎症小体类型的寡聚组装。有关此方案的使用和执行的完整详细信息，请参阅 Wu 等人。(2023)。
