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. 2019 Aug 16;9:11945. doi: 10.1038/s41598-019-48421-9

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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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A BioID screen for ligand-stimulated EphB2-proximal proteins. (a) Schematic of the BioID experiment: a FLAG-tagged biotin ligase, BirA*, was fused to the C-terminus of EphB2 and stably expressed in HEK293 cells. Depending on the presence of ephrin-B2 ligands, different proteins are recruited to the vicinity of EphB2 and labelled with biotin. (b) Western blot of biotinylation in protein lysates from our mass spec samples of HEK293 cells expressing EphB2-BirA*-FLAG (lane 2, no ligand; lane 3, 1.5 µg/mL Fc; and lane 4, 1.5 µg/mL pre-clustered ephrin-B2-Fc) or FLAG alone (lane 1) (n = 2). (c) Gene ontology and KEGG^85–87 terms associated with the proteins identified in the eB2 WD-score and Fc WD-score analysis. Blue bars represent proteins enriched in the eB2-treated samples and red bars represent proteins enriched in the Fc-treated samples. (d) Interactome webs of proteins identified in the eB2 or Fc WD-score analysis generated by Cytoscape and clustered with MCluster, divided by gene ontology term. (e) Dot plots for known EphB2 effector or trafficking-related preys in eB2 and Fc conditions. Spectral count is illustrated by fill shade, relative abundance of the protein compared to the EGFP-BirA*-FLAG condition shown by circle size, and outer circle colour represents BFDR value when compared to EGFP-BirA*-FLAG MS SAINT analysis. Full sized Western blots are in Supplementary Materials. kDa: kilodalton; eB2: ephrin-B2-Fc; BFDR: Bayesian false discovery rate.