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. 2018 Jan 31;9:463. doi: 10.1038/s41467-018-02862-4

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© The Author(s) 2018

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 — XIAP interacts with SOCS1 and enhances SOCS1 expression. a XIAP deficiency impairs IL-2-induced SOCS1 expression. T cells were collected at 0, 10, 20, 40, 60, and 120 min after IL-2 treatment. SOCS1 expression of lysate was detected by anti-SOCS1. Protein levels were quantitated by densitometry and normalized by actin control. The level of SOCS1 in WT T cells was used as 1 for comparison. b XIAP-deficiency decreases SOCS1 expression in human iTreg cells. Control and XIAP-knockdown human iTreg cells were treated with IL-2 and the levels of SOCS1 were determined at the indicated time-points. c XIAP enhances SOCS1 expression. XIAP-FLAG and SOCS1-HA were co-transfected into HEK293T cells. After 24 h of transfection, cell lysates were prepared and SOCS1 and XIAP expression was determined with anti-FLAG and anti-HA. d XIAP interacts with SOCS1. XIAP-FLAG and SOCS1-HA were co-transfected into HEK293T cells as indicated. Total cell lysates were immunoprecipitated by anti-HA and the presence of SOCS1 and XIAP-FLAG in the precipitates and lysates was determined. * indicates immunoglobulin heavy chain. e Endogenous XIAP interacts with SOCS1. Mouse peripheral T cells from spleen and lymph nodes were treated with IL-2 as indicated and then 600 μg of cell lysates were immunoprecipitated with anti-SOCS1 or control goat IgG. The contents of endogenous XIAP were determined. * indicates immunoglobulin heavy chain. f The BIR1 domain of XIAP interacts with SOCS1. Full-length (FL), RING domain-deleted (ΔR), N-terminus (N), C-terminus (C), BIR1, BIR2 or BIR3 of XIAP-FLAG were co-transfected with SOCS1-HA into HEK293T cells. Total cell lysates were immunoprecipitated with anti-HA and the presence of XIAP variants and SOCS1 in the pull-down complex and cell lysates was determined. g The SH2 domain of SOCS1 binds XIAP. Full-length (FL), SOSC box-deleted (ΔSB), N-terminal-deleted (ΔN), N-terminal (N), SH2 domain, or SOCS box (SB) of SOCS1 were transfected with XIAP-FLAG into HEK293T cells as indicated. Total cell lysates were immunoprecipitated by anti-FLAG and the presence of SOCS1 variants and XIAP in the precipitates and cell lysates was determined. Each experiment (a, c–g) was independently repeated three times with similar results