An optimized protocol to detect protein ubiquitination and activation by ubiquitination assay in vivo and CCK-8 assay
Summary: E3 ubiquitin ligases play a role in protein degradation, cellular localization, and activation, and their dysregulation is associated with human diseases. Here, we present a protocol to detect IGF2BP1 ubiquitination and activation by an E3 ubiquitin ligase FBXO45. We describe steps for prep...
| Main Authors: | , , |
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| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2023-06-01
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| Series: | STAR Protocols |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2666166723001570 |
| _version_ | 1797860178343755776 |
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| author | Xiao-Tong Lin Jie Zhang Chuan-Ming Xie |
| author_facet | Xiao-Tong Lin Jie Zhang Chuan-Ming Xie |
| author_sort | Xiao-Tong Lin |
| collection | DOAJ |
| description | Summary: E3 ubiquitin ligases play a role in protein degradation, cellular localization, and activation, and their dysregulation is associated with human diseases. Here, we present a protocol to detect IGF2BP1 ubiquitination and activation by an E3 ubiquitin ligase FBXO45. We describe steps for preparing cells and transfecting plasmids. We detail the use of western blot to detect IGF2BP1 ubiquitination and a Cell Counting Kit-8 (CCK-8) assay to detect IGF2BP1 activation. This protocol is applicable to other proteins of interest.For complete details on the use and execution of this protocol, please refer to Lin et al. (2021).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. |
| first_indexed | 2024-04-09T21:41:36Z |
| format | Article |
| id | doaj.art-c0acbd21f7084ba98fa269fb44b97111 |
| institution | Directory Open Access Journal |
| issn | 2666-1667 |
| language | English |
| last_indexed | 2024-04-09T21:41:36Z |
| publishDate | 2023-06-01 |
| publisher | Elsevier |
| record_format | Article |
| series | STAR Protocols |
| spelling | doaj.art-c0acbd21f7084ba98fa269fb44b971112023-03-26T05:19:12ZengElsevierSTAR Protocols2666-16672023-06-0142102199An optimized protocol to detect protein ubiquitination and activation by ubiquitination assay in vivo and CCK-8 assayXiao-Tong Lin0Jie Zhang1Chuan-Ming Xie2Key Laboratory of Hepatobiliary and Pancreatic Surgery, Institute of Hepatobiliary Surgery, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, ChinaKey Laboratory of Hepatobiliary and Pancreatic Surgery, Institute of Hepatobiliary Surgery, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, ChinaKey Laboratory of Hepatobiliary and Pancreatic Surgery, Institute of Hepatobiliary Surgery, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China; Corresponding authorSummary: E3 ubiquitin ligases play a role in protein degradation, cellular localization, and activation, and their dysregulation is associated with human diseases. Here, we present a protocol to detect IGF2BP1 ubiquitination and activation by an E3 ubiquitin ligase FBXO45. We describe steps for preparing cells and transfecting plasmids. We detail the use of western blot to detect IGF2BP1 ubiquitination and a Cell Counting Kit-8 (CCK-8) assay to detect IGF2BP1 activation. This protocol is applicable to other proteins of interest.For complete details on the use and execution of this protocol, please refer to Lin et al. (2021).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.http://www.sciencedirect.com/science/article/pii/S2666166723001570Cell BiologyCell CultureCell-based AssaysMolecular BiologyProtein Biochemistry |
| spellingShingle | Xiao-Tong Lin Jie Zhang Chuan-Ming Xie An optimized protocol to detect protein ubiquitination and activation by ubiquitination assay in vivo and CCK-8 assay STAR Protocols Cell Biology Cell Culture Cell-based Assays Molecular Biology Protein Biochemistry |
| title | An optimized protocol to detect protein ubiquitination and activation by ubiquitination assay in vivo and CCK-8 assay |
| title_full | An optimized protocol to detect protein ubiquitination and activation by ubiquitination assay in vivo and CCK-8 assay |
| title_fullStr | An optimized protocol to detect protein ubiquitination and activation by ubiquitination assay in vivo and CCK-8 assay |
| title_full_unstemmed | An optimized protocol to detect protein ubiquitination and activation by ubiquitination assay in vivo and CCK-8 assay |
| title_short | An optimized protocol to detect protein ubiquitination and activation by ubiquitination assay in vivo and CCK-8 assay |
| title_sort | optimized protocol to detect protein ubiquitination and activation by ubiquitination assay in vivo and cck 8 assay |
| topic | Cell Biology Cell Culture Cell-based Assays Molecular Biology Protein Biochemistry |
| url | http://www.sciencedirect.com/science/article/pii/S2666166723001570 |
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