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. Author manuscript; available in PMC: 2013 Jun 12.
Published in final edited form as: Nature. 2011 Oct 2;478(7370):529–533. doi: 10.1038/nature10509

Figure 1. A global proteomic survey identifies BET proteins as part of the PAFc and SEC.

Figure 1

(A). Proteomic strategy (B) Left: Cytoscape representation of the BET protein complex network (discussed in detail in supplementary figure 3). Bold circles indicate associations confirmed by the three orthogonal methods. Right: Heat map representing quantitative-MS data following co-IP of BETs, PAF and SEC complex members. (C) Differential proteomic analysis of the proteins interacting with I-BET and triple acetylated histone H4 tail. Left: Affinity matrices with immobilized I-BET762 or Histone H4(K5acK8acK12ac) peptide bind to the same set of BET complexes. Right: competitive inhibition of the binding of BET isoforms, and SEC and PAF complex components, to the I-BET762 matrix showing matching concentration dependence. (D) Brd4 and MLLT1 interact in HL60, MV4;11 and RS4;11 cells and binding to the I-BET762 matrix is blocked by excess I-BET151. (E) GSK1210151A (I-BET151). (F) I-BET151 binding to the acetyl-binding pocket of BRD4-BD1 (cyan) overlaid with H3K14-Acetyl peptide (green) (3jvk.pdb). A surface representation of the BRD4-BD1 is shown with key recognition and the specificity WPF shelf identified. (G) Ribbon representation of the BRD4-BD1 (cyan) crystal structure complexed with I-BET151 (shown in magenta stick format) overlaid with H3(12-19)K14ac peptide (green) taken from its complex with BRD4-BD1(3jvk.pdb). Secondary elements of the BRD4-BD1 structure have been highlighted. (H) Selectivity profile of IBET-151 showing average temperature shifts (Tm) using a fluorescent thermal shift assay. Numbering inside the spheres, e.g. 12 signifies both bromodomains 1 and 2 have been assessed. Overlaid is the selectivity profile generated using a proteomic approach (shown as boxes around proteins, discussed in Supplementary Fig. 5). Where the bromodomains have been profiled by both thermal shift and proteomic approaches the agreement is excellent. Proteins not assessed by either technique are shown in grey. (I) Comparison of I-BET762 and I-BET151 potency in ligand displacement assays, direct BIAcore binding and LPS stimulated IL6 cytokine production from human PBMCs or whole blood (WB).