Knockdown of HDAC6 expression inhibits binding of ATP to hsp90 and affects the binding of hsp90 to its cochaperone p23. (A) K562 cells expressing HDAC6 siRNA or K562 vector control cells were treated with the indicated dose of 17-AAG for 16 hours. After this treatment, cell lysates were affinity precipitated with ATP-Sepharose and immunoblotted with anti-hsp90 antibody. The levels of hsp90 after 17-AAG treatment are as indicated. (B) Vector control K562 and HDAC6 siRNA knockdown cells were left untreated or treated for 16 hours with 3 and 5 μM 17-AAG. Lysates were immunoprecipitated with anti-p23 antibody followed by immunoblotting with anti-hsp90 and anti-p23 antibody. (C) Alternatively, lysates were immunoprecipitated with anti-cdc37 antibody followed by immunoblotting with anti-hsp90 and anti-cdc37 antibody.