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. 2018 Dec 5;9:5200. doi: 10.1038/s41467-018-07258-y

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — BD2 of BRD4 recognizes and interacts with K242/245 di-acetylated FOXO3a. a HA-tagged FOXO3a was co-expressed with Flag-tagged BD1 and BD2 in HEK293 cells treated with 2 μM TSA and 4 mM Nicotinamide overnight. After IP with anti-HA antibody, FOXO3a acetylation and its association with BD1 and BD2 were examined by western blotting using pan acetylated-lysine and anti-Flag antibodies. b Wild-type (WT) or K242R/245 R mutant (KR) of FOXO3a were co-expressed with Flag-tagged BRD4 in HEK293 cells treated with 1 μM MK2206 for 2 day. After IP with anti-HA antibody, FOXO3a acetylation and its association with BRD4 were examined by western blotting using pan acetylated-lysine and anti-Flag antibodies. c WT or KR mutant of FOXO3a was co-expressed with Flag-tagged BD2 in HEK293 cells treated with 2 μM TSA and 4 mM Nicotinamide overnight. After IP with anti-HA antibody, FOXO3a acetylation and its association with BD2 were examined by western blotting. d The FOXO3a-binding selectivity of the BD1 and BD2 of BRD4. 2D 1H-15N HSQC spectra of BRD4-BD1 or BD2 in the free form (black) and in the presence of a FOXO3a peptide (residues 238–249, NPDGG-K242ac-RG-K245ac-APR) that is non-acetylated, or contains single or di-acetylated K242 or K245 with protein:ligand molar ratio of 1:5 (red). Other FOXO members were also included for analyses. e Left and middle panel: Stereo ribbon diagram of the 3D solution structure of the BRD4-BD2 bound to a di-acetylated K242ac/K245ac FOXO3a peptide (cyan). Side chains of key residues engaged at the protein/peptide interactions are depicted and color-coded by atom type. Right panel: surface-filled representation of the 3D structure of the BRD4-BD2 highlights its same mode of ligand recognition for FOXO3a (cyan) and Twist (yellow) peptides. f FOXO3a was co-expressed with full-length WT- or HN-BRD4 in HEK293 cells treated with 1 μM MK2206 for 2 day. After FOXO3a and BRD4 were immunoprecipitated, the associated BRD4 and FOXO3a were examined by western blotting