title: IP-FCM platform detects the existence and regulator-caused dissociation of components in naturally assembled HSP90 complex.
abstract: 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.
authors.affiliation: Sino-French Cooperative Central Lab, Shanghai Gongli Hospital, Pudong New District, Shanghai, People's Republic of China.
authors.forename: Fan-Fan
authors.initials: FF
authors.lastname: Cao
authors.forename: Li-Min
authors.initials: LM
authors.lastname: Xu
authors.forename: Xue
authors.initials: X
authors.lastname: Zhang
authors.forename: Ying
authors.initials: Y
authors.lastname: Wang
authors.forename: Mao-Quan
authors.initials: MQ
authors.lastname: Li
authors.forename: Georges
authors.initials: G
authors.lastname: Uzan
authors.forename: Bin
authors.initials: B
authors.lastname: Peng
authors.forename: Deng-Hai
authors.initials: DH
authors.lastname: Zhang
mesh_terms: D015153:Blotting, Western; D045744:Cell Line, Tumor; D005434:Flow Cytometry / Q000379:methods*; D018841:HSP90 Heat-Shock Proteins / Q000032:analysis*; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods*; D008863:Microspheres
chemical_list: D018841:HSP90 Heat-Shock Proteins
keywords: 17-N-allylamino-17-demethoxygeldanamycin; Cdc37; HSP70; celastrol; flow cytometry; heat shock protein 90 (HSP90); polystyrene microsphere