Identification of phosphorylation site on PARP1 mediating its cytosolic translocation in virus-infected HeLa cells

Fei Wang; Ming Tong Ma; Junfang Xu; Haipeng Liu

doi:10.1016/j.xpro.2022.101808

Identification of phosphorylation site on PARP1 mediating its cytosolic translocation in virus-infected HeLa cells

STAR Protoc. 2022 Nov 2;3(4):101808. doi: 10.1016/j.xpro.2022.101808. eCollection 2022 Dec 16.

Authors

Fei Wang¹, Ming Tong Ma², Junfang Xu³, Haipeng Liu⁴

Affiliations

¹ Shanghai 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. Electronic address: wangfei_tj@qq.com.
² Shanghai 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.
³ Clinical Translation Research Center, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai 200433, China.
⁴ Shanghai 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; Central Laboratory, Shanghai Pulmonary Hospital, School of Medicine, Tongji University School of Medicine, Shanghai 200433, China. Electronic address: haipengliu@tongji.edu.cn.

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).

Keywords: Cell biology; Cell separation/fractionation; Mass spectrometry; Microscopy; Molecular biology; Signal transduction.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Cytosol
HeLa Cells
Humans
Phosphorylation
Poly (ADP-Ribose) Polymerase-1
Translocation, Genetic*

Substances

PARP1 protein, human
Poly (ADP-Ribose) Polymerase-1