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目的:探讨氧化应激对热休克蛋白90α(Hsp90α)与ADP-核糖基化因子1(ARF1)细胞内定位、相互作用的影响。方法:应用500μM H2O2处理HepG2细胞,建立氧化应激模型,MTT比色法检测细胞活力,Western blotting检测Hsp90α和ARF1水平,细胞免疫荧光法、免疫共沉淀检测上述蛋白在氧化应激下的分布、共定位变化和相互作用。结果:MTT比色法结果提示,随氧化应激时间延长,细胞存活力降低;Western blotting结果显示,氧化应激可提高胞内Hsp90α和ARF1蛋白水平;免疫共沉淀结果显示,随氧化应激作用时间延长,Hsp90α与ARF1相互结合增多;细胞免疫荧光结果显示,随氧化应激作用时间延长,Hsp90α与ARF1荧光强度增强,并趋于沿胞膜分布。结论:提示氧化应激影响Hsp90α和ARF1的水平、胞内分布及相互作用。
Objective: To investigate the effect of oxidative stress on the localization and interaction of heat shock protein 90α (Hsp90α) and ADP-ribosylation factor 1 (ARF1). Methods: HepG2 cells were treated with 500μM H2O2 to establish a model of oxidative stress. Cell viability was measured by MTT colorimetric assay. Hsp90α and ARF1 were detected by Western blotting. Immunofluorescence and immunoprecipitation were used to detect the distribution of these proteins under oxidative stress. Colocalization changes and interactions. Results: The results of MTT assay indicated that the cell viability decreased with the prolongation of oxidative stress. Western blotting showed that oxidative stress increased intracellular levels of Hsp90α and ARF1 protein. Co-immunoprecipitation showed that with oxidative stress Hsp90α and ARF1 increased with the time prolonging. The results of immunofluorescence showed that the fluorescence intensities of Hsp90α and ARF1 increased along with the prolongation of oxidative stress, and they tended to distribute along the membrane. Conclusion: It suggests that oxidative stress affects the levels, intracellular distribution and interactions of Hsp90α and ARF1.