Supplementary Figure 9: Chromatin recruitment experiments employing recombinant Brd4 constructs and DNL-2.

(a) Resin-bound Brd4-BD1-BD2 was incubated with DNL-2. All subsequent steps were performed as in Fig. 2c. Internal normalization: H4Kac₅ variant, set as 1 (red asterisks). The experiment was performed in duplicate, and the resulting data is plotted employing the same grid as in Supplementary Fig. 8c. (b) Left: Schematic representation of His₆-Brd4-BD1-Flag construct and PAGE analysis of the protein, which was expressed recombinantly in E.coli and purified via Nickel-NTA and size exclusion chromatography. Right: Resin-bound Brd4-BD1 was incubated with DNL-2. All subsequent steps were performed as in Fig. 2c. Internal normalization: H4Kac₅ variant, set as 1 (red asterisks). The experiment was performed in duplicate. (c) Left: Schematic representation of His₆-Brd4-BD2-Flag, expressed and purified identically as described in (b). Right: His₆-Brd4-BD2-Flag was incubated with DNL-2. All subsequent steps were performed as in Fig. 2c, except that no internal normalization was performed. The input (IN)-normalized reads are shown from 2 independent pulldown (PD) experiments. (d) Analysis of recombinant full-length human p300 constructs. N-terminally Flag-tagged full-length human p300 constructs were expressed recombinantly in Sf9 cells, purified on M2 anti-Flag affinity matrix and analyzed by denaturing PAGE followed by CBB staining (lane 1: Y1089A/F1090S; lane 2: ΔBD; lane 3: wt).