CAJ | 학술논문

目的：探究鼠双微体2同源体（MDM2）与端粒保护蛋白1（POT1）在细胞水平是否有相互作用，及其是否发挥E3泛素化连接酶的功能。方法：首先，用蛋白酶体抑制剂MG132处理稳定表达Flag-POT1的HeLa细胞，Western印迹检测Flag-POT1的表达情况；其次，在HeLa细胞中转入外源的Myc-MDM2和Flag-POT1质粒，48 h后收集细胞，通过免疫共沉淀方法验证Myc-MDM2和Flag-POT1是否具有相互作用；再次，在稳定表达Flag-POT1的HeLa细胞中转入Myc-MDM2或MDM2 siRNA，48 h后收集细胞，Western印迹检测Flag-POT1的表达水平。结果：MG132处理后， Flag-POT1的表达量明显升高且有拖尾现象，免疫共沉淀显示Myc-MDM2和Flag-POT1具有相互作用，但无论转入Myc-MDM2还是MDM2 siRNA，Flag-POT1的表达水平没有明显变化。结论：POT1通过泛素化途径降解，MDM2与POT1具有相互作用，但MDM2不是POT1主要的E3泛素化连接酶。
목적：탐구서쌍미체2동원체（MDM2）여단립보호단백1（POT1）재세포수평시부유상호작용，급기시부발휘E3범소화련접매적공능。방법：수선，용단백매체억제제MG132처리은정표체Flag-POT1적HeLa세포，Western인적검측Flag-POT1적표체정황；기차，재HeLa세포중전입외원적Myc-MDM2화Flag-POT1질립，48 h후수집세포，통과면역공침정방법험증Myc-MDM2화Flag-POT1시부구유상호작용；재차，재은정표체Flag-POT1적HeLa세포중전입Myc-MDM2혹MDM2 siRNA，48 h후수집세포，Western인적검측Flag-POT1적표체수평。결과：MG132처리후， Flag-POT1적표체량명현승고차유타미현상，면역공침정현시Myc-MDM2화Flag-POT1구유상호작용，단무론전입Myc-MDM2환시MDM2 siRNA，Flag-POT1적표체수평몰유명현변화。결론：POT1통과범소화도경강해，MDM2여POT1구유상호작용，단MDM2불시POT1주요적E3범소화련접매。
Objective: To investigate whether there was interaction between mouse double minute 2 homolog (MDM2) and protection of telomeres(POT1), and whether POT1 was degradated by MDM2 as an E3 ubiquitin li-gase. Methods: Firstly, HeLa cells with overexpression of Flag-POT1 were treated with proteasome inhibitor MG132, then Flag-POT1 level was detected by Western blot. Secondly, HeLa cells transfected with Myc-MDM2 and Flag-POT1 plasmids were incubated for 48 h before harvest, then MDM2 and POT1 interaction was identified by immunoprecipitation. Thirdly, Myc-MDM2 and MDM2 siRNA were transfected into HeLa cells with overexpres-sion of Flag-POT1, POT1 level were detected by Western blot. Results: The expression level of Flag-POT1 in-creased after treatment with MG132, immunoprecipitation test demonstrated the interaction between POT1 and MDM2, there was no obvious expression change of Flag-POT1 with or without Myc-MDM2 or MDM2 siRNA. Con-clusion: POT1 was degradated through the ubiquitin pathways, there was interaction between MDM2 and POT1, and MDM2 was not an E3 ubiquitin ligase for POT1.