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. 2022 May 9;29(11):2203–2217. doi: 10.1038/s41418-022-01010-2

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© The Author(s) 2022

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. 2 — A Immunoaffinity purification and mass spectrometry analysis of RUNX2-containing protein complexes. Whole-cell extracts from MDA-MB-231 cells stably expressing FLAG-Vector or FLAG-RUNX2 were immunopurified using anti-FLAG affinity columns and eluents with FLAG peptide. Elutes were resolved using SDS-PAGE and silver-stained. Protein bands were retrieved and analyzed using mass spectrometry. B Western blotting analysis of the purified fractions using antibodies against FLAG in MDA-MB-231 cells. C Immunoprecipitation with antibody against RUNX2 followed by immunoblotting with antibodies against MTA1, MTA2, MTA3, HDAC1, HDAC2, RbAp46/48, MBD3, CUL4B, DDB1, and ROC1. D Immunoprecipitation with antibodies against MTA1, MTA2, MTA3, HDAC1, HDAC2, RbAp46/48, MBD3, CUL4B, DDB1, and ROC1 followed by immunoblotting with antibody against RUNX2. E, F GST pull-down assays with GST-fused proteins expressed in bacteria and in vitro translated proteins as indicated. G–I Identification of the essential domains of RUNX2, MTA1, or CUL4B required for interaction. J Schematic diagram depicting molecular interactions among RUNX2/NuRD(MTA1)/CRL4B complex. IB, immunoblotting; aa, amino acid.