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. 2011 Nov 21;208(12):2545–2560. doi: 10.1084/jem.20110853

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© 2011 Kim et al.

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Figure 1. — IGSF4 is expressed in human and mouse T cells and positively regulates T cell responses. (A) Western blot and RT-PCR analyses. (top) Tissue distribution of IGSF4 in 8-wk-old mice. IGSF4 expression in T cell lines (bottom left) and purified mouse or human T cell subsets (bottom right). #, clone number; SP, splenic CD3⁺ T cells. (B) Jurkat T cells (top), human PBLs (bottom left), and mouse splenic CD3⁺ T cells (bottom right) were stimulated for the indicated times with anti-CD3/28, and the expression of IGSF4 or IL-2 was assessed by quantitative PCR and flow cytometric analyses (top), RT-PCR analysis (bottom left), and Western blot analysis (bottom right). (A and B) The data are representative of three independent experiments. (C) Jurkat T cells (left) and human PBLs (middle) were transfected with either 70 µM of scrambled (SC) siRNA or siRNA targeting IGSF4. IGSF4 expression was measured after 48 h of transfection (top). Stimulation of cells and IL-2 mRNA measurements were performed as described in B. The results are the mean ± SD of triplicate experiments. (right) Purified splenic CD3⁺ T cells from TCR transgenic DO11.10 mice were transfected with 1 µg/100 µl shRNA targeting IGSF4. The IGSF4 knockdown efficiency was analyzed after 48 h of transfection (top). The cells were coincubated with 50 ng/ml OVA–pulsed A20 mouse B cells, and the IL-2 mRNA levels were measured. The results are the mean ± SD of triplicate experiments. (D, top) Establishment of Jurkat T cells overexpressing GFP or WT-IGSF4_GFP by lentiviral transduction. The efficiency of viral infection was determined by Western blot and flow cytometric analyses. IB, immunoblot. (bottom) The cells were then stimulated with SEE-pulsed Raji B cells, anti-CD3/28, or PMA/A23187 for the indicated time. IL-2 mRNA levels were assessed by RT-PCR (blots) and real-time quantitative PCR (graphs) analyses. The results are the mean ± SD of triplicate experiments. Molecular mass (M) is indicated in kilodaltons. EV, empty vector.