DEK interacts preferentially with dephosphorylated C/EBPα. (A) Expression of C/EBPα–FLAG/HA and endogenous C/EBPα proteins in the chromatin fraction of MOLM-14 cells after doxycycline withdrawal for 14 hours and subsequent treatment with MEK inhibitor PD 98059 for 6 hours. TATA-binding protein (TBP) served as the loading control. (B) Detection of S21 phosphorylation on C/EBPα–FLAG/HA immunoprecipitated from the chromatin compartment 6 hours after treatment with MEK inhibitor. Representative quantification data (iTRAQ reporter ion signals) corresponding to DEK peptide (C; EPFTIAQGK) and C/EBPα peptide (D; VLELTSDNDR) after treatment with MEK inhibitor, immunoprecipitation of C/EBPα, and proteomics analysis, as described in supplemental Methods and supplemental Figure 2. (E) Detection of DEK protein after immunoprecipitation of C/EBPα–FLAG/HA from the chromatin fraction of MOLM-14 cells subjected to doxycycline induction and treatment with MEK inhibitor. Western blots against DEK and C/EBPα–FLAG/HA in total cell extracts served as a control for each immunoprecipitation. (F) Western blot for C/EBPα after immunoprecipitation of endogenous DEK from MOLM-14 cells treated with MEK inhibitor. Immunoblot against TBP served as a loading control. (G) Coimmunoprecipitation of C/EBPα–FLAG/HA WT and mutants S21A and S21D (designated as WT, S/A and S/D) by DEK-V5 in 293T cells. Western blots were performed against V5 and HA, with TBP probed as a loading control.