Acetylation of lysine 121 and 198 regulates C/EBPε activity. (A) COS cells were transfected with Flag-C/EBPε together with HA-p300 and treated with NAM. C/EBPε protein bands were isolated from the lysates and processed for mass spectrometry analysis. Data represent the identified lysines and functional domain location. (B-C) COS cells were transfected with Flag-C/EBPε single-lysine knock-out (from WT) and “addback” (from lysine dead) together with HA-p300, followed by Flag immunoprecipitation. Cell lysates were analyzed by WB, using anti-acetylated lysines (AcK) and anti-Flag antibody. (D) M-CSFR promoter luciferase activity was analyzed in COS cells transfected with Flag-C/EBPε, Flag-C/EBPε K121R, Flag-C/EBPε K198R, Flag-C/EBPε K121/198R, and C/EBPε K15xR. An empty vector plasmid was used as control. All values were normalized for cotransfected renilla. (E) Lactoferrin reporter activity was analyzed in COS cells transfected with Flag-C/EBPε, Flag-C/EBPε K121/198R, or C/EBPε K15xR. Cell lysates were analyzed for C/EBPε expression by WB, using an anti-Flag antibody. (F) Nuclear lysates were prepared from COS cells transfected with Flag-C/EBPε, Flag-C/EBPε K121R, Flag-C/EBPε K198R, and Flag-C/EBPε K15xR, followed by pull-down of biotinylated oligonucleotides. Cell lysates were analyzed by WB, using anti-Flag antibody. As controls, oligonucleotides representing a mutated binding site were used. IP, immunoprecipitation; WB, western blot; WCL, whole-cell lysate. Data (B-F) are representative of 3 or more independent experiments; error bars represent standard error of the mean (between experiments). *P < .05; **P < .01.