Protocol for quantification of the lysosomal degradation of extracellular proteins into mammalian cells
Summary: 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 wi...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2021-12-01
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| Series: | STAR Protocols |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S266616672100681X |
| _version_ | 1819174899446448128 |
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| author | Ayaka Tomihari Momoka Chiba Akira Matsuura Eisuke Itakura |
| author_facet | Ayaka Tomihari Momoka Chiba Akira Matsuura Eisuke Itakura |
| author_sort | Ayaka Tomihari |
| collection | DOAJ |
| description | Summary: 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). |
| first_indexed | 2024-12-22T20:46:18Z |
| format | Article |
| id | doaj.art-73c2926b026747bab84edd04ce386c97 |
| institution | Directory Open Access Journal |
| issn | 2666-1667 |
| language | English |
| last_indexed | 2024-12-22T20:46:18Z |
| publishDate | 2021-12-01 |
| publisher | Elsevier |
| record_format | Article |
| series | STAR Protocols |
| spelling | doaj.art-73c2926b026747bab84edd04ce386c972022-12-21T18:13:13ZengElsevierSTAR Protocols2666-16672021-12-0124100975Protocol for quantification of the lysosomal degradation of extracellular proteins into mammalian cellsAyaka Tomihari0Momoka Chiba1Akira Matsuura2Eisuke Itakura3Department of Biology, Graduate School of Science and Engineering, Chiba University, Chiba, JapanDepartment of Biology, Graduate School of Science and Engineering, Chiba University, Chiba, JapanDepartment of Biology, Graduate School of Science, Chiba University, Inage-ku, Chiba, JapanDepartment of Biology, Graduate School of Science, Chiba University, Inage-ku, Chiba, Japan; Corresponding authorSummary: 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).http://www.sciencedirect.com/science/article/pii/S266616672100681XCell BiologyFlow Cytometry/Mass CytometryCell-based AssaysMolecular/Chemical ProbesProtein expression and purification |
| spellingShingle | Ayaka Tomihari Momoka Chiba Akira Matsuura Eisuke Itakura Protocol for quantification of the lysosomal degradation of extracellular proteins into mammalian cells STAR Protocols Cell Biology Flow Cytometry/Mass Cytometry Cell-based Assays Molecular/Chemical Probes Protein expression and purification |
| title | Protocol for quantification of the lysosomal degradation of extracellular proteins into mammalian cells |
| title_full | Protocol for quantification of the lysosomal degradation of extracellular proteins into mammalian cells |
| title_fullStr | Protocol for quantification of the lysosomal degradation of extracellular proteins into mammalian cells |
| title_full_unstemmed | Protocol for quantification of the lysosomal degradation of extracellular proteins into mammalian cells |
| title_short | Protocol for quantification of the lysosomal degradation of extracellular proteins into mammalian cells |
| title_sort | protocol for quantification of the lysosomal degradation of extracellular proteins into mammalian cells |
| topic | Cell Biology Flow Cytometry/Mass Cytometry Cell-based Assays Molecular/Chemical Probes Protein expression and purification |
| url | http://www.sciencedirect.com/science/article/pii/S266616672100681X |
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