Department of Biology, Graduate School of Science and Engineering, Chiba University, Chiba, Japan.
Department of Biology, Graduate School of Science, Chiba University, Inage-ku, Chiba, Japan.
STAR Protoc. 2021 Nov 23;2(4):100975. doi: 10.1016/j.xpro.2021.100975. eCollection 2021 Dec 17.
Endocytic internalization of extracellular proteins plays roles in signaling, nutrient uptake, immunity, and extracellular protein quality control. However, there are few protocols for analyzing the lysosomal degradation of extracellular protein. Here, we purified secreted proteins fused with pH-sensitive GFP and acid- and protease-resistant RFP from mammalian cells and describe an internalization assay for mammalian cells. This protocol enables quantification of cellular uptake and lysosomal degradation of protein-of-interest (POI) via cell biological and biochemical analyses. For full details on the use and execution of this protocol, please refer to Itakura et al. (2020).
细胞外蛋白的内吞内化在信号转导、营养物质摄取、免疫和细胞外蛋白质量控制中发挥作用。然而，目前用于分析细胞外蛋白溶酶体降解的方法并不多。在这里，我们从哺乳动物细胞中纯化了与 pH 敏感 GFP 和酸及蛋白酶抗性 RFP 融合的分泌蛋白，并描述了一种用于哺乳动物细胞的内化测定方法。该方案可通过细胞生物学和生化分析来定量测定目的蛋白（POI）的细胞摄取和溶酶体降解。有关该方案使用和实施的详细信息，请参阅 Itakura 等人（2020 年）。
