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Updated: Aug 21, 2025
Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay
Published on: May 3, 2018
Identification of phosphorylation site on PARP1 mediating its cytosolic translocation in virus-infected HeLa cells
Fei Wang1, Ming Tong Ma1, Junfang Xu2
1Shanghai Key Laboratory of Tuberculosis, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai 200433, China; Department of Microbiology and Immunology, Tongji University School of Medicine, Shanghai 200072, China; Clinical Translation Research Center, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai 200433, China.
Abstract:
Poly (ADP-ribose) polymerase 1 (PARP1) localization is controlled by its phosphorylation state. Here, we describe a protocol to monitor PARP1 subcellular localization in HSV-1-infected HeLa cells using immunofluorescence microscopy and cytoplasmic/nuclear fractionation. We detail steps to identify phosphorylation sites on PARP1 using conserved motif analysis and mass spectrometry. This protocol can be applied to the study of other protein phosphorylation events in other cell types. For complete details on the use and execution of this protocol, please refer to Wang et al. (2022).
Insights
This study details a protocol to track Poly (ADP-ribose) polymerase 1 (PARP1) in infected cells. It also outlines methods for identifying PARP1 phosphorylation sites, aiding future research on protein regulation.
Area of Science:
- Molecular Biology
- Cell Biology
- Virology
Background:
- Protein localization is crucial for cellular function.
- Phosphorylation significantly influences protein activity and localization.
- Poly (ADP-ribose) polymerase 1 (PARP1) is a key enzyme involved in DNA repair and other cellular processes.
Purpose of the Study:
- To establish a protocol for monitoring PARP1 subcellular localization in HSV-1 infected HeLa cells.
- To identify specific phosphorylation sites on PARP1.
- To provide a versatile method applicable to other proteins and cell types.
Main Methods:
- Immunofluorescence microscopy to visualize PARP1.
- Cytoplasmic and nuclear fractionation to separate cellular compartments.
- Conserved motif analysis and mass spectrometry for phosphorylation site identification.
Main Results:
- A detailed protocol for tracking PARP1 localization in infected cells was successfully developed.
- Key phosphorylation sites on PARP1 were identified.
- The protocol demonstrated applicability across different cell types and for studying other phosphorylation events.
Conclusions:
- The developed protocol enables precise monitoring of PARP1 subcellular localization.
- Understanding PARP1 phosphorylation is critical for elucidating its regulatory mechanisms.
- This methodology serves as a valuable tool for broader research in cell signaling and protein regulation.
