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. 2021 Jun 11;12(6):608. doi: 10.1038/s41419-021-03887-2

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© The Author(s) 2021

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. 5 — A Interaction analysis between DYRK1A and SPRY2 in the presence of WT TRAF2 or its truncates. V5-SPRY2 was co-transfected with Flag-DYRK1A, Myc-TRAF2, or its different truncations into HEK293T cells and Flag-affinity purified. Immunoprecipitates were probed with α-Flag and α-V5 to analyze the enrichment of SPRY2 in DYRK1A pulldowns after overexpression of TRAF2 and truncates. B Analysis of phosphorylation status of SPRY2 in the presence of DYRK1A and TRAF2. V5-SPRY2 was co-transfected with Flag-DYRK1A (WT, PVQE/AAAA or K188R) and/or Myc-TRAF2 (WT, ΔRING or ΔTRAF) in HEK293T cells, followed by V5-affinity purification. Immunoprecipitates were probed with α-phospho-pan-Ser/Thr antibody. Full-length TRAF2, but not ΔRING or ΔTRAF deletions, was able to promote phosphorylation of SPRY2 in the presence of wild-type DYRK1A. Also, Flag-DYRK1A-PVQE/AAAA mutant did not promote the phosphorylation of SPRY2. C Cellular localization of V5-SPRY2, Myc-TRAF2, Flag-DYRK1A, and Rab5. HEK293T cells were co-transfected with V5-SPRY2, Flag-DYRK1A, and/or Myc-TRAF2, and immunostained with respective antibodies. Images were acquired using ZEISS 710 confocal laser scanning microscope. The first three panels, scale bar = 25 μm. The last panel, scale bar = 1 μm.