Validation of new Hsp90 clients. (A) Network analysis of a selected set of potentially new Hsp90 client proteins. stSILAC data and the Hsp90 interaction network Hsp90Int (Echeverria et al, PlosOne 2011) were combined to identify interesting candidates (OGT, ITK and BRAT1) with no reported interactions with Hsp90 at the time of the analysis. Edges connecting candidate proteins with known Hsp90 interacting proteins are highlighted in red. (B) Co-immunoprecipitation (co-IP) experiment demonstrating interactions between BRAT1, OGT and ITK with Hsp90b in Jurkat cells. Equal concentrations of specific antibodies against BRAT1 (rabbit), OGT (rabbit), ITK (mouse), Hsp90b (mouse) and the corresponding non-immune control antibodies from rabbit and mouse were used in co-IP experiments, and then analyzed by immunoblotting (WB). (C) GA-induced degradation of BRAT1, ITK and OGT in Jurkat cells. Lysates from cells treated with GA or with the equivalent volume of the solvent DMSO (control) for 6 and 20 hs were analyzed by WB for these three mentioned proteins and also for Hsp70 and CDK6 as positive controls of GA action.