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. 2019 Apr 25;9:6539. doi: 10.1038/s41598-019-42990-5

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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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Workflow and strategy for generation of endogenous DYRK1A interactome. (A) Schematic overview of fractionation and IPMS strategy. HeLa S3 cells were grown in 6 L flasks before harvesting at 1 million cells/ml. Large scale fractionation of cell pellets generated protein lysates representing a cytosolic, nuclear and chromatin bound fractions. Subsequent immunoprecipitations were done in triplicate prior to sample preparation for mass spectrometry identification. (B) Domain arrangement of DYRK1A and regions containing antibody epitope as reported by manufacturers. NLS, nuclear localization signal; DH, DYRK homology box; PEST, proline glutamic acid serine and threonine rich sequence; His, polyhistidine stretch; S/T serine and threonine rich region. Epitopes recognized by each antibody used for IP experiments fall within the designated amino acid region and represented by black bar. (p) rabbit polyclonal; (m) mouse monoclonal. (C) Number of novel and literature reported HCI’s by antibody. (Abnova n = 3; Abcam n = 3; Bethyl n = 3; SC: Santa Cruz n = 3) FC-A > 3; SAINT > 0.7. (D) Overlap between HCI’s of each set of immunoprecipitations by antibody.