Figure 2

Nutlin-induced modulation of acetylated proteins in the AML cell line MOLM-13. (A) MOLM-13 cells were subjected to stable isotope labeling with amino acids in cell culture (SILAC). Cells were labeled with either light (L-Lysine-2HCl, L-Arginine-HCl) or heavy (¹³C₆ L-Lysine-2HCl, ¹³C₆¹⁵ N₄ L-Arginine-HCl) isotopes of amino acids and treated with DMSO (control) or 6 μM nutlin-3, respectively, for 6 hours. Cells were harvested and lysed, and lysates were mixed at a ratio of 1:1 (5 mg protein of each). The lysate was precleared with uMACs protein G Microbeads, then precleared with beads and an unspecific antibody (rabbit IgG), before immunoprecipitation of acetylated proteins using an anti-acetyl-lysine antibody and Microbeads. Proteins were eluted in 95°C SDS loading buffer and subjected to one-dimensional gel electrophoresis (SDS-PAGE) and staining with Coomassie Blue. Bands were excised and peptides generated by trypsination. Peptides were separated and fragmented using LC-MS/MS (LC-LTQ-Orbitrap) and protein ID’s and H/L ratios were obtained using MaxQuant and Perseus software. (B) Representative MS/MS spectrum of peptides derived from Histone H2B.