Validation of nutlin-induced modulation of acetylated proteins in the AML cell line MOLM-13. (A) Western blots of total lysates from SILAC (stable labeling with amino acids in cell culture) labeled MOLM-13 cells treated with DMSO or 6 μM nutlin-3 for 6 hours using antibodies against p53, MDM2, Histone H2B, acetylated Histone H2B (Lys120), Hsp27, Hsp90, acetylated Hsp90 (Lys294) and actin. Bands were quantified using region of interest imaging analysis, and values are given as fold induction of control relative to actin. (B) MOLM-13 cells were treated nutlin-3 as described above, and immunoprecipitations using anti-acetyl lysine antibody or an unspecific antibody (rabbit IgG) were performed. Western blots of Hsp27 of the immunoprecipitated proteins and of the total lysates used in immunoprecipitations are shown. (C) Total levels of Hsp90 and Hsp27 in MOLM-13 cells treated with nutlin-3 as described above analyzed by flow cytometry. Results are given as representative flow diagrams and median fluorescence intensity (MFI) relative to control.