Treatment with PS or AUY922 depletes the binding of CXCR4 and GRK3 with hsp90 in AML cells. (A) HEK293/CXCR4 cells were treated with the indicated concentrations of PS for 8 hours. After treatment, cell lysates were prepared and Myc-tagged CXCR4 and hsp90 were immunoprecipitated. Immunoblot analyses were performed for Myc tag and hsp90 on the immunoprecipitates and total cell lysates. The expression levels of β-actin in the lysates served as the loading control. (B) OCI-AML3 cells were treated with the indicated concentrations of PS for 8 hours. At the end of treatment, cells were harvested and CXCR4 was immunoprecipitated from the total cell lysates. Immunoblot analyses were performed for CXCR4, hsp90, and hsp70 on the immunoprecipitates. Immunoblot analyses were also performed for acetylated K69 hsp90 and hsp70 in the total cell lysates. The expression levels of β-actin in the lysates served as the loading control. (C) OCI-AML3 cells were treated with the indicated concentrations of bortezomib (BZ) and PS for 8 hours. Then, total cell lysates were prepared and immunoblot analyses were performed for CXCR4 and c-RAF on the total cell lysates. The expression levels of β-actin in the lysates served as the loading control. (D) OCI-AML3 cells were treated with the indicated concentrations of AUY922 for 8 hours. At the end of treatment, cells were harvested and CXCR4 and hsp90 were immunoprecipitated from the total cell lysates. Immunoblot analyses were performed for CXCR4, hsp90, and hsp70 on the immunoprecipitates. (E) OCI-AML3 cells were treated with the indicated concentrations of PS or AUY922 for 4 hours. After treatment, hsp90 and GRK3 were immunoprecipitated from the total cell lysates. Immunoblot analyses were performed for GRK3 and hsp90 on the immunoprecipitates.