Preliminary screening of novel IER5 interacting proteins after ionizing irradiation
10.3760/cma.j.issn.0254-5098.2017.04.002
- VernacularTitle:宫颈癌细胞放射损伤后IER5蛋白表达及其相互作用蛋白的筛选和验证
- Author:
Xinping YU
;
Teng MA
;
Pingkun ZHOU
;
Yumei WU
- Keywords:
Cervical cancer;
Immediate early response 5;
Immuniprecipitation;
Mass spectrometry
- From:
Chinese Journal of Radiological Medicine and Protection
2017;37(4):246-250
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate the expression of radiation-induced IER5 protein and screen its potential interaction proteins that may participate in DNA repair process.Methods HeLa cells were irradiated with 4 Gy ionizing radiation.IER5 protein expression in whole cell lysate and in nuclear fraction were detected by Western blot at different timepoints after irradiation.3 × Flag-IER5 pCMV plasmid was constrcuted and the Flag tagged-IER5 expression was verified by Western blot.293T cells were transfected with 3 × Flag-IER5 pCMV plasmid.After irradiation the cells were collected and proteins were extracted.The IER5 interaction proteins were purified using immunoprecipitation and separated by 12％ SDS-polyacrylamide gel electrophoresis.Then the binding proteins were cut from the gel and analyzed by Mass spectrometry.Results The expression of IER5 protein began to increase 4 hour post-irradiation and its peak level was observed at 12 hour post-irradiation,and it lasted until 48 hour after irradiation.The expression level of IER5 protein in whole cell lysate and nuclear fraction were both increased.With the mass spectrometry analysis,a total of 347 proteins and 256 proteins were identified in irradiated and nonirradiated groups,respectively.Fourty one differential proteins were obtained,where 10 proteins were associated with DNA metabolic process and DNA rapair in the irradiated group and the poly(ADP-ribose) polymerase 1 (PARP1) protein was further confirmed by Western blot.Conclusions IER5 protein is an DNA damage related protein,and it may participate in DNA repair process.
