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. 2019 Sep 5;17(3):319–331. doi: 10.1016/j.gpb.2018.12.010

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© 2019 The Authors

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

PMC Copyright notice

Identification of protein binders for BETi JQ1 by using PLATO-BC assay

A. Rank of ORF enrichment from the PLATO-BC assays for biotinylated JQ1 (Bio-JQ1) relative to free biotin (Bio). 12,492 ORFs were ranked according to the ratio of Bio-JQ1/Bio (from the smallest to the largest). The red line shows the cut-off value. A known protein target of JQ1, BRD2, was ranked as a top hit (red dot). B. Hit validation of JQ1 protein targets identified from the PLATO-BC assay. pET-DEST42-EWSR1/EYA3/RBM14/BD1/BD2 was transformed into the E. coli DE3 strain. V5-His6-tagged EWSR1/EYA3/RBM14/BD1/BD2 proteins were purified and subjected to protein pull-down assays for Bio-JQ1 or Bio. Protein samples were separated by SDS-PAGE and analyzed by immunoblotting using an anti-V5 antibody. C. Interaction of EWSR1 with H4ac peptide in vitro. V5-His6-tagged EWSR1 protein was purified and subjected to protein pull-down assays for biotinylated, acetylated or non-acetylated H4 (Bio-H4ac or Bio-H4) peptide. Protein samples were separated by SDS-PAGE and analyzed by immunoblotting using an anti-V5 antibody. D. Interaction of EWSR1 with H4ac in cells. pcDNA-DEST40-EWSR1 was transiently transfected in HEK293T cells. Cell lysate was prepared and subjected to the protein immunoprecipitation assays for V5-His6-tagged EWSR1 using an anti-V5 mouse antibody or a mouse IgG (mIgG) control. Protein samples were separated by SDS-PAGE and analyzed by immunoblotting using an anti-H4ac antibody. E. JQ1 interrupts the interaction of EWSR1 with H4ac peptide in vitro. V5-His6-tagged EWSR1 protein was purified and incubated with JQ1 (at 1 μM or 10 μM) or DMSO, prior to the protein pull-down assays for Bio-H4ac or free biotin (Bio). Protein samples were separated by SDS-PAGE and analyzed by immunoblotting using an anti-V5 antibody or HRP-conjugated streptavidin to detect EWSR1 or H4ac respectively. BETi, bromodomain and extraterminal domain inhibitor; BRD2, bromodomain containing 2; H4ac, acetylated histone H4; EWSR1, Ewing sarcoma breakpoint region 1; EYA3, EYA transcriptional coactivator and phosphatase 3; RBM14, RNA-binding protein 14; BD1, bromodomain 1; BD2, bromodomain 2.