C/EBPε acetylation is regulated by SIRT1 and p300. (A) COS cells were transfected with HA-C/EBPε and treated overnight in the absence or presence of 500 μM (+), 1 mM (++) VPA, 1 mM (+), 5 mM (++) NAM, followed by HA immunoprecipitation. Cell lysates were analyzed by WB, using anti-acetyl lysines (AcK) and anti-HA antibodies. (B) COS cells were cotransfected with HA-C/EBPε and Myc-SIRT1, followed by HA immunoprecipitation. Cell lysates were analyzed by WB, using anti-AcK, anti-HA, and anti-Myc antibodies. (C) COS cells were cotransfected with Flag-C/EBPε and HA-p300, followed by Flag immunoprecipitation. Cell lysates were analyzed by WB, using anti-AcK, anti-Flag, and anti-HA antibodies. (D) COS cells transfected with FLAG-C/EBPε and HA-p300 or Myc-SIRT1, followed by PLA. Cells were probed for Flag, HA, or Myc. Single transfections and pMT2 empty vector were used as controls. PLA signal (red dots) represents colocalization of C/EBPε with p300 and SIRT1. (E) CD34⁺ cells were differentiated toward neutrophils. At day 10, cells were treated overnight with 5 mM NAM, followed by AcK immunoprecipitation. Cell lysates were analyzed by WB, using an anti-C/EBPε antibody. (F) Day 10 neutrophil precursors were isolated and treated overnight with NAM, followed by PLA. Cells were probed with anti-AcK and anti-C/EBPε antibodies. PLA signal (red dots) represents C/EBPε acetylation. IP, immunoprecipitation; NAM, nicotinamide; VPA, valproic acid; WB, western blot; WCL, whole-cell lysate. Data are representative of 3 or more independent experiments.