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. Author manuscript; available in PMC: 2014 May 6.
Published in final edited form as: Nat Immunol. 2010 Aug 8;11(9):862–871. doi: 10.1038/ni.1917

Figure 4.

Figure 4

The intracellular CYT-1 domain of CD46 and SPAK are required for IL-10 production in CD4+ T cells. (a) Secretion of IL-10 by Jurkat T cells stably transfected with the BC1 isoform (Jurkat-BC1) or BC2 isoform (Jurkat-BC2) of CD46 and either not activated or activated for 48 h with anti-CD3 and anti-CD46 or with anti-CD46 alone. (b) Immunoprecipitation (with a CD46-specific mAb or isotype-matched control mAb) of lysates of primary human CD4+ T cells activated for 15 min with various antibodies (above lanes), followed by immunoblot analysis with anti-CD46 (top) or anti-SPAK (bottom). Left margin: CD46, BC1 + BC2, CD46 isoform with BC region and either CYT-1 or CYT-2; CD46, C1 + C2, CD46 isoform with only the C region. Lysate (far left lane), untreated lysates from nonactivated cells (control). (c) IL-10 secretion by purified CD4+ T cells left untransfected (NT) or transfected with SPAK-specfic siRNA, nonspecific siRNA (Control) or buffer alone (Mock) and then either not activated or activated for 36 h with mAb to CD3 alone or mAb to CD3 and mAb to CD46 (key). (d) IL-10 production by Jurkat T cells left untransfected (Jurkat) or stably transfected with wild-type SPAK (Jurkat-SPAK) or a ‘kinase-dead’ version of SPAK (Jurkat-ΔSPAK) and either not activated or stimulated with anti-CD3 and anti-CD46 or with anti-CD46 alone. *P < 0.01 (Student’s t-test). Data are representative of three experiments (mean ± s.d. in a,c,d).