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. 2019 Feb 13;33(8):2006–2021. doi: 10.1038/s41375-019-0403-2

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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. 2 — IKZF1 and IKZF3 utilize the N-terminal ZnF domain to associate with the activation and inhibition domain of RUNX1. a HEK293T cells were transfected with constructs encoding an empty vector (EV), FLAG-tagged IKZF3 wild-type (WT), or mutants. A schematic representation of IKZF3 mutants is shown. IKZF3 mutants that interact (+) or do not interact (−) with RUNX1 are shown. b Immunoblot analysis of FLAG-IKZF3 immunoprecipitation (IP). Immunocomplexes were probed with antibodies to the indicated proteins. c HEK293T cells were transfected with constructs encoding an empty vector (EV), FLAG-tagged IKZF3 wild-type (WT) or mutants as indicated. The cell extracts were subjected to anti-FLAG IP and the immunocomplexes were probed with antibodies to the indicated proteins. d Purified GST-tagged proteins as indicated were incubated with in vitro translated FLAG-tagged RUNX1. GST pull-downs were probed with anti-FLAG antibodies. Ponceau S staining shows the expressions of GST-proteins. e Schematic representation of RUNX1 mutants. RUNX1 mutants that interact (+) or do not interact (−) with IKZF3 are shown. f Immunoblot analysis of GST pull-downs. Purified GST-tagged proteins as indicated were incubated with in vitro translated FLAG-tagged RUNX1. GST pull-downs were probed with anti-FLAG antibodies. Ponceau S staining shows the expressions of GST-proteins. g Same as in f, except that the indicated GST-proteins were used. h HEK293T cells stably expressing IKZF3 were transfected with constructs encoding an empty vector (EV), FLAG-tagged RUNX1 wild-type (WT), or mutants as indicated. The whole cell lysates (WCL) were subjected to anti-FLAG IP and the immunocomplexes were probed with antibodies to the indicated proteins. i Schematic model of the interaction between IKZF1/3 and the AD and ID of RUNX1. Unless otherwise noted, immunoblots are representative of three independent experiments