Analysis of the binding of KU174 to Hsp90. Drug Affinity Responsive Target Stability (DARTS) was used to test the specificity of KU174 to Hsp90. Recombinant Hsp90 was incubated with 25 μM of KU174, 17-AAG, radicicol or vehicle, followed by digestion with thermolysin and analysis by SDS-PAGE Western blot for protection of Hsp90 protein (Figure 5A). Upper band is protected from proteolysis by both N-terminal and C-terminal Hsp90 inhibitors. Biotinylated KU174 (b-KU174) but not the inactive analogue (-noviose) bound with sufficient affinity to immunoprecipitate Hsp90 in native PC3-MM2 cell lysates (Figure 5B). Importantly, binding was prevented with excess ATP. KU174 was injected over Hsp90β immobilized to the surface of a SPR sensor chip at concentrations of 0.25, 0.5, 1.0, 10, 50, 100, and 200 μM as described under "Materials and Methods". Sensorgrams of KU174 binding to Hsp90β are shown (Figure 5C, left) and a concentration dependent binding curve for the interaction of KU174 with Hsp90β (Figure 5C, right).