Native Size Exclusion Chromatography (SEC) fractionation of Hsp90 complexes and inhibition of Hsp90 dependent luciferase refolding activity. Vehicle or 25 μM KU174 treated (24h) SEC fractions were probed by SDS-PAGE Western blot. For each protein in Figure 4A, a vehicle control treated (Figure 4A, panel C) and a KU174 treated (Figure 4A, panel T) are depicted. Pooled vehicle fractions (9-16) showed the ability to refold thermally denatured luciferase and this activity was inhibited by KU174 (Figure 4B). In a follow-up study, PC3-MM2 cells treated with or without KU174 (0.1 μM) were fractionated by SEC and fractions 9-16 of each sample were pooled and chaperone activity was tested as a function of luciferase refolding (Figure 4C). The chaperone refolding activity of 0.1 μM KU174 treated samples is reduced to ~50% of vehicle (see left axis segment) and was further inhibited by novobiocin. These data suggest KU174 directly inhibits Hsp90 complex refolding activity.