Cao Fan-Fan, Xu Li-Min, Zhang Xue, Wang Ying, Li Mao-Quan, Uzan Georges, Peng Bin, Zhang Deng-Hai
Sino-French Cooperative Central Lab, Shanghai Gongli Hospital, Pudong New District, Shanghai, People's Republic of China.
Cytometry A. 2014 Apr;85(4):359-67. doi: 10.1002/cyto.a.22421. Epub 2013 Nov 21.
Flow cytometry, in conjunction with immunoprecipitation (IP-FCM), is suggested to have some advantages to conventional IP-western blot technology in analyzing protein complexes. In this paper, to further examine its practicability, we test the use of IP-FCM in detecting the HSP90 complex, which has gained importance in drug research and development and involves more than a dozen components. We found that IP-FCM could effectively detect HSP70, p23, Cdc37, and Cdk6 components in the HSP90 complex naturally formed in U937 cells when this complex was captured by anti-HSP90 antibody-coated polystyrene microspheres. IP-FCM could also detect alteration in components caused by treating cells with HSP90 inhibitors. In a cell-free environment, IP-FCM could detect the direct effects of ATP and/or HSP90 inhibitors (17-N-allylamino-17-demethoxygeldanamycin or celastrol) in causing component dissociation and the time- and dose-effects of inhibitor-caused dissociation. IP-FCM is a practical and powerful platform for analyzing HSP90 complex components, and is thus a useful tool in studying HSP90 complex function and screening inhibitors.
流式细胞术结合免疫沉淀法（IP-FCM），在分析蛋白质复合物方面，相较于传统的免疫沉淀-蛋白质印迹技术具有一些优势。在本文中，为进一步检验其实用性，我们测试了IP-FCM在检测HSP90复合物中的应用，该复合物在药物研发中具有重要意义且涉及十几种成分。我们发现，当用抗HSP90抗体包被的聚苯乙烯微球捕获U937细胞中自然形成的HSP90复合物时，IP-FCM能够有效检测其中的HSP70、p23、Cdc37和Cdk6成分。IP-FCM还能检测用HSP90抑制剂处理细胞后复合物成分的变化。在无细胞环境中，IP-FCM能够检测ATP和/或HSP90抑制剂（17-N-烯丙基氨基-17-去甲氧基格尔德霉素或雷公藤红素）导致成分解离的直接作用以及抑制剂引起解离的时间和剂量效应。IP-FCM是分析HSP90复合物体成分的实用且强大的平台，因此是研究HSP90复合物功能和筛选抑制剂的有用工具。
