Supplementary Figure 8: Chromatin recruitment experiments employing recombinant histone mark readers.
From: Accelerated chromatin biochemistry using DNA-barcoded nucleosome libraries
(a) Top: Schematic representation of the recombinant Flag-tagged Brd4 construct, spanning BD1 and BD2. Bottom: Flag-Brd4-BD1-BD2 was expressed in Sf9 cells and purified by anti-Flag immunoprecipitation. The purity of the protein was assessed by denaturing PAGE followed by CBB staining. (b) DNL-1 incubation of resin-bound Flag-Brd4-BD1-BD2 identical to Fig. 2c (replicate 2). (c) Combinations of histone modifications (‘mod’) selected for the second version of the library (‘DNL-2’). Unmodified (‘–mod’) or mono-/di-/hyperacetylated H3 proteins (horizontal axis) were combined with otherwise unmodified histones (‘–mod’) or mono-/hyperacetylated H4 (vertical axis). (d) Analysis of the combined DNL-2 by native gel electrophoresis and ethidium bromide (EtBr) DNA staining. (e) Incubation of resin-bound GST-tagged p300-BD-PHD with DNL-2. All subsequent steps were performed as in Fig. 2b. Internal normalization: H4Kac5 variant, set as 1 (red asterisk). Experiment was performed in duplicate.
