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. 2022 Jun 6;13(6):528. doi: 10.1038/s41419-022-04984-6

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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. 3 — a Immunoprecipitation with anti-Flag antibody of protein extracts from HEK293T cells co-transfected with Flag-tagged EphA2 and V5-tagged EGFR. Western blot analysis was carried out with the indicated antibodies. b Immunoprecipitation with anti-Flag antibody of extracts from HEK293T cells co-transfected with Flag-tagged Ephexin1, V5-tagged EGFR, and/or V5-tagged EphA2 plasmids. Western blot analysis was carried out with the indicated antibodies. c Immunoprecipitation with anti-Flag antibody of extracts from HEK293T cells transfected with Flag-tagged Ephexin1 and treated with EGF (100 ng/ml) after 16 h of serum starvation. Western blot analysis was carried out with the indicated antibodies. d Proximity ligation assay (PLA) of EphA2 and EGFR in either shControl or shEphexin1-H1299 cells showing the location of both proteins (red). The cells were counterstained with DAPI (blue) to visualize the nuclei. Scale bar = 10 um. e Quantification of the Proximity ligation assay (PLA) shown in (d). Data are shown as mean ± SD. ***P < 0.001 as determined through a two-tailed Student’s t test. f, g Lysates from shControl or shEphexin1 H1299 cells were immunoprecipitated with anti-EGFR antibody or anti-EphA2 antibody. western blot analysis with the indicated antibodies. H Immunoprecipitation analysis with anti-Flag antibody and analysis with the indicated antibodies of HEK293T cells co-transfected with Flag-tagged EphA2, V5-tagged EGFR, and/or V5-tagged Ephexin1 plasmids. I Pulldown assays using an anti-Flag antibody of the Flag-tagged EphA2_Kinase domain with GST or GST-Ephexin1. j Pulldown assays using an anti-Flag antibody of the Flag-tagged-EphA2_kinase domain, HA-tagged-EGFR_RR domain, and GST-Ephexin1 as indicated.