HCT116 cells were seeded in 10-cm cell culture dishes to reach ~90% confluence. Then, the cells were incubated with the indicated concentrations of compounds or the same amount of DMSO for 12 hours. After washing the cells twice with ice-cold PBS, radioimmunoprecipitation assay (RIPA) buffer [50 mM tris (pH 7.4), 150 mM NaCl, 1% NP-40, and protease inhibitor cocktail (Roche)] was used to lyse the cells for 1 hour on ice. Cell lysates were centrifuged at 12,000 rpm at 4°C for 15 min. Then, 1 mg of cell lysates was incubated with 5 μg of anti-Hsp90 (sc-13119, Santa Cruz Biotechnology), anti-Cdc37 (4793S, Cell Signaling Technology), or normal rabbit immunoglobulin G (IgG) (sc-2027, Santa Cruz Biotechnology) separately and overnight at 4°C on a vertical roller. Protein A/G Magnetic Agarose Beads (78609, Thermo Fisher Scientific) were added to lysates for another 3-hour incubation at 4°C. Last, the beads were washed five times with RIPA buffer and subjected to SDS-PAGE, followed by Western blot analysis.

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