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. 2017 Jun 9;8:15732. doi: 10.1038/ncomms15732

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Copyright © 2017, The Author(s)

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,b) Immunoblot analysis (IB) of HEK293 cells co-transfected with Flag-tagged full-length Tespa1 (FL) or different Tespa1 mutants and HA-tagged IP3R1-N-TM. Cells were lysed and immunoprecipitated (IP) with anti-Flag beads (a) or anti-HA antibodies plus protein G beads (b), then probed with anti-Flag and anti-HA antibodies. Bottom, immunoblot analysis of whole-cell lysates (WCL, without immunoprecipitation). (c) Immunoblot analysis of Jurkat cells transfected with Flag-tagged Tespa1 (J-Tespa1), Flag-tagged Tespa1-F187A/F188A (J-FFAA) or Flag empty vector (J-Vector). Cells were left unstimulated (0) or were stimulated with anti-CD3 and anti-CD28 antibodies for 5 or 10 min, lysed, immunoprecipitated with anti-Flag beads and probed with anti-IP3R1, anti-IP3R2 or anti-IP3R3 antibodies. Data are representative of at least three experiments.