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.

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.