Fig. 3
From: IL-6 receptor blockade corrects defects of XIAP-deficient regulatory T cells
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
