In a cellular context, Hsp90 acts in multiprotein complexes. Therefore, interfering with Hsp90 function may lead to the disruption of these complexes. In a cellular assay, western blotting was performed under reducing (+dithiothreitol or +DTT) and non-reducing (-DTT) conditions after 5b incubation of the K562 cells. Similarly to AX, 28 5b inhibited the formation of Hsp90 higher-order multimeric species, in contrast to novobiocin (NB), but in concordance with AUY922 (SI Figure ). Next, to study the effect of 5b exposure on Hsp90 native multiprotein complexes, blue native (BN) PAGE analysis was carried out with K562 cell lysates after 5b incubation. At cytotoxic concentrations of 5b, Hsp90ɑ and Hsp90β multiprotein complexes were disrupted, including monomers/dimers of Hsp40 and Hsp27; Hsp60 (primarily in mitochondria) multiprotein complexes, serving as a loading control, were not affected (Figure ).