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. 2016 Feb 5;2(2):e1501257. doi: 10.1126/sciadv.1501257

Fig. 3. E2F-1 interacts with BRD4 on cytokine gene promoters.

Fig. 3

(A) Heat map of bromodomain binding to acetylated E2F-1 peptides (sequence: 113-RHPGKGVKSPGEKSR-127). The bromodomains are structurally subgrouped into eight families (I to VIII) (21), and the multiple domains of a given bromodomain protein are represented by numbers in brackets. (B) Interaction between HA–E2F-1 and Flag-BD1/BD2 in human embryonic kidney (HEK) 293T cells transfected with HA–E2F-1 (1 μg) and Flag-BD1 or Flag-BD2 (2 μg) (BD 1 and BD 2 of BRD4, respectively). Flag-agarose beads were used for immunoprecipitation. (C) Interaction between E2F-1 and BRD4 in DMSO-differentiated HL60 cells. E2F-1 (C20) or rabbit IgG antibodies were used for immunoprecipitation. (D to I) qPCR double ChIP analysis of BRD4/E2F-1 interaction in DMSO-differentiated HL60 cells challenged with LPS (100 ng/ml, 3 hours) (D), treated with JQ1 (5 μM, 4 hours) (E and F), or BB-Cl-amidine (2.5 μM, 16 hours) (G), with accompanying immunoblot ± SD (H and I). *P < 0.05, **P < 0.01. (J) Interaction between His-BD1 and E2F-1 from U2OS cells treated with JQ1 (5 μM) or BB-Cl-amidine (5 μM) for 16 hours. The cell lysates were incubated with His-tagged BD1 of BRD4 and subject to Ni-agarose immunoprecipitation.