Cotreatment with LBH589 and 17-AAG causes greater attenuation of the levels of Bcr-Abl and FLT-3, and inhibits STAT5 DNA binding in K562 and MV4-11 cells. (A) Following treatment with 10 nM of LBH589 and/or 1000 nM 17-AAG for 24 hours, Western blot analyses of p21, Bcr-Abl, p-STAT5, STAT5, Bcl-x_L, Pim-2, p-AKT, AKT, p-ERK1/2, ERK1/2, and PARP were performed on the cell lysates from K562 cells. The levels of β-actin served as the loading control. (B) Following treatment with 5 nM of LBH589 and/or 100 nM 17-AAG for 24 hours, Western blot analyses of p21, p-FLT-3, FLT-3, p-AKT, AKT, p-ERK1/2, ERK1/2, p-STAT5, STAT5, and PARP were performed on the cell lysates from MV4-11 cells. The levels of β-actin served as the loading control. (C,D) Cotreatment with LBH589 and/or 17-AAG inhibits STAT5 DNA binding activity more than either agent alone in K562 (C) and MV4-11 cells (D). Following treatment of K562 or MV4-11 cells with indicated concentrations of LBH589 and/or 17-AAG for 24 hours, nuclear extracts were tested for the DNA binding activity of STAT5 by EMSA. For supershift analysis, the nuclear extracts were treated with anti-STAT5 antibody before the addition of the poly(dI-dC) and the labeled DNA probe.