| Wu Chenglin,Liu Xiaodan,Wang Yuxiao,Du Li,Fu Kaifei,Zhou Lijun.Screening and verifying the proteins interacting with phosphorylation cluster of DNA-PKcs by yeast two-hybrid assay[J].Chinese Journal of Radiological Medicine and Protection,2017,37(6):401-407 |
| Screening and verifying the proteins interacting with phosphorylation cluster of DNA-PKcs by yeast two-hybrid assay |
| Received:March 06, 2017 |
| DOI：10.3760/cma.j.issn.0254-5098.2017.06.001 |
| KeyWords:Yeast two-hybrid DNA-PKcs Phosphorylation cluster Interaction Co-Immunoprecipitation (Co-IP) |
| FundProject:国家自然科学基金（31470897，81530085） |
| Author Name | Affiliation | E-mail | | Wu Chenglin | Central Laboratory, Navy General Hospital, Beijing Key Laboratory for Radiobiology, Beijing 100850, China | | | Liu Xiaodan | Institute of Radiation Medicine, Academy of Military Medical Sciences, Beijing Key Laboratory for Radiobiology, Beijing 100850, China | | | Wang Yuxiao | Central Laboratory, Navy General Hospital, Beijing Key Laboratory for Radiobiology, Beijing 100850, China | | | Du Li | Institute of Radiation Medicine, Academy of Military Medical Sciences, Beijing Key Laboratory for Radiobiology, Beijing 100850, China | | | Fu Kaifei | Central Laboratory, Navy General Hospital, Beijing Key Laboratory for Radiobiology, Beijing 100850, China | | | Zhou Lijun | Central Laboratory, Navy General Hospital, Beijing Key Laboratory for Radiobiology, Beijing 100850, China | hzzhoulj@126.com |
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| Abstract:: |
| Objective To screen and verify the proteins interacting with phosphorylation cluster of DNA dependent protein kinase catalytic subunit ((DNA-PKcs) by yeast two-hybrid assay. Methods To know the proteins interacting with DNA-PKcs phosphorylation cluster,yeast two-hybrid assay was applied to screen the cDNA library of human hepatic tissue with a previously constructed plasmid pGBKT7-DPC.The positive clones were further identified by PCR,rotary validation and sequence analysis.Then the eukaryotic expression vectors of the bait protein and screened positive clone proteins were constructed and transfected into human embryonic kidney 293T cells to detect whether the proteins could been expressed correctly.At last,the bait protein and screened positive clone proteins were co-transfected into 293T cells and protein interaction was detected with Co-Immunoprecipitation (Co-IP) assay. Results After two rounds of screening using the yeast two-hybrid assay,12 candidate clones were obtained.Then 7 clones with different insert fragments were identified by PCR,and 3 positive proteins interacted with DNA-PKcs phosphorylation cluster were further verified by rotary validation.Sequencing analysis demonstrated that these 3 proteins were MBNL1,SIK2 and YY1AP1,respectively.Accordingly,the eukaryotic expression vectors of bait protein and 3 positive clone proteins were constructed successfully and expressed correctly in 293T cells.Finally,the Co-IP assay confirmed that these 3 positive clone proteins could interact with DNA-PKcs phosphorylation cluster. Conclusions Proteins interacting with DNA-PKcs phosphorylation cluster are successfully screened and identified. |
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