Figure 3.

Specificity of 5b against Hsp90 CTD and its cochaperone function. (a) Recombinant (full-length) Hsp90α (1 μg) was incubated with 5b at indicated concentrations, followed by digestion with thermolysin. Treated protein samples were electrophoresed (SDS-PAGE) and immunoblotted with anti-Hsp90α for detecting the protection of Hsp90α protein by 5b (the upper band is protected from proteolysis). (b) A cell-free thermal shift assay was performed by incubating recombinant Hsp90α CTD protein with 5b at an increasing temperature (up to 95 °C). The melting temperature (T_m) without inhibitors (DMSO) was used as a control. (c) Dose-dependent intracellular (K562 cells) thermal stabilization (CETSA_ITDRF) of Hsp90 after 5b incubation (24 h) at its increasing concentration (1.25–5 μM). (d) 5b inhibits the Hsp90α chaperone function, comparable to TM and GM, in the cell-free luciferase refolding assay, where the incubation of the inhibitors prevented the rabbit reticulocyte lysate (a source of Hsp90)-assisted refolding of denatured luciferase. (e) Incubation of 5b blocked the binding of Hsp90 CTD-interacting cochaperone (PPID) in TR-FRET measurements. (f) 5b did not reduce the amount of Hsp90-bound FITC-labeled GM and, therefore, does not compete for the GM binding pocket of full-length Hsp90α. Unlabeled GM, GP, PUH71, and TM served as positive controls and NB and CA1 as negative controls.