HL-60/LR cells are collaterally sensitive to 17-AAG. (A) HL-60 and HL-60/LR cells were treated with the indicated concentrations of 17-AAG for 48 hours. After this, the cells were stained with trypan blue and the percentage of positively stained nonviable cells was determined by light microscopy. Values on the curves represent the mean plus or minus SE of 2 experiments performed in duplicate. (B) HL-60 and HL-60/LR cells were treated with the indicated concentrations of 17-AAG for 24 hours. After this Western blot analyses were done for p-AKT, AKT, p-ERK1/2, ERK1/2, c-Raf, and hsp70 on the cell lysates. The levels of β-actin expression served as the loading control. (C) Reduced binding of ATP and p23 to hyperacetylated hsp90 in HL-60/LR versus HL-60 cells. After incubation of cell lysates containing hsp90 with ATP-sepharose, ATP-sepharose was pelleted and Western blot analysis was done for hsp90. Alternatively, immunoprecipitates were probed with anti-p23 and anti-hsp90 antibody. (D) After treatment of HL-60 and HL-60/LR cells with 17-AAG, lysates were immunoprecipitated with anti-Bax (6A7) antibody and immunoblotted with anti-Bcl_XL or anti–total Bax antibody.