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. 2019 May 13;10:2146. doi: 10.1038/s41467-019-09981-6

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — EZHIP forms a stable complex with PRC2 and lowers H3K27me3 in vivo. a Immunoblots of whole cell lysates generated from 293T cells expressing HA-FLAG-tagged H3.3 WT or K27M/R or FLAG-tagged EZHIP WT or R405E mutant. b Schematic showing the strategy for purification of EZHIP-associated proteins from 293T cells. c Silver stain of FLAG-tagged EZHIP association with PRC2. d Immunoprecipitated material from c were subjected to mass-spectrometry for protein identification and quantification. Proteins identified with at least 2 unique peptides and a log₂ fold-change over the mock negative control of greater than 4 were considered hits. Protein abundances were calculated using ProteomeDiscoverer software. Complete list is provided as Supplementary Dataset 2. e Silver stained SDS-PAGE gel of mono-Q column fractions of M2 eluate from c. f, g Immunoblots displaying co-fractionation of EZHIP and PRC2 subunits on mono-Q and Superdex 200 columns, respectively. h Sequence alignment displaying the conserved 12 amino acid sequence in the EZHIP C-terminus. Red and blue domains represent the EZHIP conserved sequence and the site of hotspot mutations in PFA ependymomas respectively. i Immunoblots of lysates generated from mouse embryonic fibroblasts with EED knockout or expressing human EZHIP WT, H3K27M, or H3K27R. j Immunoblots of lysates generated from human 293T cell lines expressing human EZHIP WT or D81Y or mouse EZHIP. k Immunoblots of 293T expressing EZHIP WT, R405E, R407E or R405A;R407A mutants. Source images for Immunoblots are provided in a source data file