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Figure 3. KDM2B forms a variant PRC1 complex in mouse ESCs containing RING1B and PCGF1.(A) To purify KDM2B and associated proteins, a mouse ESC cell line stably expressing Flag-2XStrepII-tagged KDM2B was generated. Nuclear extract was isolated from this cell line, KDM2B affinity purified, and the purified proteins subject to mass spectrometry. (B) Purified KDM2B fractions were resolved by gradient SDS-PAGE and visualized by SyproRuby staining. The purifications were performed in the absence and presence of benzonase to exclude DNA-mediated interactions and a cell line containing only the empty vector was used to control for non-specific binding to the affinity matrix. The elutions were probed by western blot for KDM2B as indicated. (C) Elutions from the KDM2B affinity purification were directly analysed by tryptic digestion followed by peptide identification by LC–MS/MS. The Mascot scores and peptide coverage are shown for the respective affinity purifications. KDM2B in ESCs associates with a variant PRC1 complex containing RING1B, BCOR/BCORL1, PCGF1, RYBP, YAF2 and SKP1. (D) Western blot analysis of endogenous KDM2B immunoprecipitation from ESC nuclear extract, verifying that KDM2B interacts with RING1B, but not the PRC2 component EZH2. Flag immunoprecipitation was performed as negative control. (E) Reciprocal affinity purifications and subsequent LC-MS/MS for RING1B, YAF2, RYBP, and PCGF1 confirm the interaction between KDM2B and these PRC1 components. This analysis further indicates that PCGF1 is unique to the KDM2B-PRC1 complex. Protein identification scores and sequence coverage (Cov [%]) are indicated. (F) A schematic representation of the variant KDM2B PRC1 complex (left panel) in comparison to canonical PRC1 complexes (right panel).DOI: http://dx.doi.org/10.7554/eLife.00205.007Figure 3—figure supplement 1. KDM2B forms a variant PRC1 complex in mouse ESCs containing RING1B and PCGF1.(A)–(D) Stable cell lines expressing Flag-2XStrepII tagged RING1B (A), YAF2 (B), RYBP (C), and PCGF1 (D) were generated and each protein affinity purified from mouse ESC nuclear extract. Purified fractions were resolved by gradient SDS-PAGE and visualized by SyproRuby staining. In each case the input, flowthrough, and elution is indicated above. The elutions were probed by western blot against the tagged protein as indicated below each SyproRuby stained gel.DOI: http://dx.doi.org/10.7554/eLife.00205.008 Image published in: Farcas AM et al. (2012) Copyright © 2012, Farcas et al. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View

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