Depletion of HDAC6 increases the affinity of acetylated hsp90 for 17-AAG. (A) K562 cells were transfected with control vector or siRNA to HDAC6. After 48 hours, the cells were harvested and lysed. The lysates were incubated with or without indicated doses of 17-AAG for 30 minutes at 4°C, and then incubated with biotin-GM for 1 hour at 4°C. To this, washed Streptavidin-agarose beads were added and incubated overnight at 4°C. The immunoprecipitates were washed and proteins were eluted with SDS sample loading buffer before the immunoblot analyses with specific antibody against hsp90. Bands in the Western blots were quantified by densitometry using ImageQuant5.2 (GE Healthcare, Piscataway, NJ). (B) Vector of siHDAC6-transfected cells were exposed to indicated dose of 17-AAG after 48 hours of transfection. Hsp90 acetylation was assessed as described in “Acetylation of Hsp90 and its binding to ATP-Sepharose.” Vertical line has been inserted to indicate a repositioned lane from the same gel.