中华放射医学与防护杂志
中華放射醫學與防護雜誌
중화방사의학여방호잡지
Chinese Journal of Radiological Medicine and Protection
2013年
3期
235-238
,共4页
朱翔%曹宝山%张照辉%马力文%苏旭%梁莉
硃翔%曹寶山%張照輝%馬力文%囌旭%樑莉
주상%조보산%장조휘%마력문%소욱%량리
辐射损伤%转化生长因子β激酶1%多聚泛素化%NF-κB%信号通路
輻射損傷%轉化生長因子β激酶1%多聚汎素化%NF-κB%信號通路
복사손상%전화생장인자β격매1%다취범소화%NF-κB%신호통로
Radiation effect%Transforming growth factor-β-activating kinase 1%Polyubiquitination%Nuclear factor-kappa B%Signaling pathway
目的 研究转化生长因子激酶1(TAK1) Lys 63连接多聚泛素化在辐射导致的NF-κB激活过程中起到的作用及其作用位点.方法 通过基因瞬时转染技术将FLAG-TAK1和HA-Ub-K63质粒共同转染至HEK-293T细胞,应用免疫沉淀和免疫印迹技术研究辐射对TAK1泛素化的影响；用稳定转染TAK1表达的HeLa细胞,证实辐射对TAK1泛素化的影响.通过免疫印迹技术,比较分析辐射对稳定转染TAK1 K158R的MEF细胞与TAK1野生型MEF细胞比较分析辐射对它表达IKKs、p38及JNK的影响,以证明辐射诱导的TAK1-Lys 63多聚泛素化的位点是否在TAK1赖氨酸158位点.结果 瞬时转染的HEK-293T细胞接受辐射后1h开始出现明显的TAK1泛素化表现,2h更明显.在稳定表达FLAG-TAK1的HeLa细胞系中得到证实.与TAK1野生型MEF细胞相比,TAK1 K158R突变的MEF细胞中辐射诱导的IKKs和p38磷酸化作用减低.结论 TAK1多聚泛素化在辐射导致的NF-κB激活过程中起到关键作用,辐射所致TAK1-Lys 63连接多聚泛素化发生在TAK1的赖氨酸158位点.
目的 研究轉化生長因子激酶1(TAK1) Lys 63連接多聚汎素化在輻射導緻的NF-κB激活過程中起到的作用及其作用位點.方法 通過基因瞬時轉染技術將FLAG-TAK1和HA-Ub-K63質粒共同轉染至HEK-293T細胞,應用免疫沉澱和免疫印跡技術研究輻射對TAK1汎素化的影響；用穩定轉染TAK1錶達的HeLa細胞,證實輻射對TAK1汎素化的影響.通過免疫印跡技術,比較分析輻射對穩定轉染TAK1 K158R的MEF細胞與TAK1野生型MEF細胞比較分析輻射對它錶達IKKs、p38及JNK的影響,以證明輻射誘導的TAK1-Lys 63多聚汎素化的位點是否在TAK1賴氨痠158位點.結果 瞬時轉染的HEK-293T細胞接受輻射後1h開始齣現明顯的TAK1汎素化錶現,2h更明顯.在穩定錶達FLAG-TAK1的HeLa細胞繫中得到證實.與TAK1野生型MEF細胞相比,TAK1 K158R突變的MEF細胞中輻射誘導的IKKs和p38燐痠化作用減低.結論 TAK1多聚汎素化在輻射導緻的NF-κB激活過程中起到關鍵作用,輻射所緻TAK1-Lys 63連接多聚汎素化髮生在TAK1的賴氨痠158位點.
목적 연구전화생장인자격매1(TAK1) Lys 63련접다취범소화재복사도치적NF-κB격활과정중기도적작용급기작용위점.방법 통과기인순시전염기술장FLAG-TAK1화HA-Ub-K63질립공동전염지HEK-293T세포,응용면역침정화면역인적기술연구복사대TAK1범소화적영향；용은정전염TAK1표체적HeLa세포,증실복사대TAK1범소화적영향.통과면역인적기술,비교분석복사대은정전염TAK1 K158R적MEF세포여TAK1야생형MEF세포비교분석복사대타표체IKKs、p38급JNK적영향,이증명복사유도적TAK1-Lys 63다취범소화적위점시부재TAK1뢰안산158위점.결과 순시전염적HEK-293T세포접수복사후1h개시출현명현적TAK1범소화표현,2h경명현.재은정표체FLAG-TAK1적HeLa세포계중득도증실.여TAK1야생형MEF세포상비,TAK1 K158R돌변적MEF세포중복사유도적IKKs화p38린산화작용감저.결론 TAK1다취범소화재복사도치적NF-κB격활과정중기도관건작용,복사소치TAK1-Lys 63련접다취범소화발생재TAK1적뢰안산158위점.
Objective To study the role of the transforming growth factor-β-activating kinase 1 (TAK1) poly-ubiquitination in radiation-induced NF-κB activation.Methods FLAG-TAK1 and HA-UbK63 plasmids were transiently transfected into HEK-293T cells.The radiation effect on TAK1 ubiquitination was detected by immunoprecipitation and Western blot,which was further confirmed in FLAG-TAK1 HeLa stable cell line.Results At 1 h after irradiation,Lys 63-1ink TAK1 ubiquitination was induced in the HEK-293T cells with plasmids transfection.This result was further confirmed by using a FLAG-TAK1 stably expressed HeLa cell line.Radiation-induced IKKs and p38 phosphorylation were greatly impaired in MEFs reconstituted with TAK1 K158R mutant compared to ones with wild-type TAK1.Conclusions Lys63-1inked TAK1 poly-ubiquitination at Lys-58 plays a key role in the process of radiation-induced NF-κB activation.
