HTRF assays were performed using a standard protocol ( A total volume of 8 μl of His-tag Hsp90 and GST-tag Cdc37 was premixed in PBS buffer containing an additional 200 mM KF (pH 7.4), with a final concentration of 80 nM. Then, 4 μl of prediluted solution with compounds at the indicated concentration was added and incubated for 1 hour at 37°C. Subsequently, 4 μl of anti-GST Cryptate (61GSTKLA, Cisbio) and 4 μl of anti-6HisXL665 (61HISXLA, Cisbio) detection reagent were added to reach a 20-μl volume for the experimental system, which was then incubated for another 30 min at room temperature. Time-resolved fluorescence intensities were measured using a Molecular Devices instrument (SpectraMax Paradigm; excitation, 320 nm; emission, 665 and 620 nm). The final HTRF ratio was calculated by taking the ratio of signals at two different wavelengths as follows: ratio = (Signal 665 nm/Signal 620 nm) × 10,000.

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