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Figure S1. Generation of Stx11-/- mice. (A) Structure of the targeted region of the murine Stx11 gene, the targeting vector, and the deleted gene construct resulting from homologous recombination. The murine Stx11 gene consists of one coding exon which was completely deleted. The two homology regions are indicated as gray dashed boxes adjacent to the coding exon. The probe used for Southern blot analysis of genomic DNA digested with HindIII is indicated as a black line. The location of the neomycin resistance gene (Neo) and one copy of the diphteria toxin gene (dta) in the targeting vector are shown. (B) Southern blot analysis of genomic DNA from different Stx11 genotypes. Genomic DNA was digested with HindIII, separated electrophoretically, blotted onto a nylon membrane, and probed with the radioactively labelled probe indicated in A. The detected bands represent the WT, or the knockout allele. (C) Western blot analysis for the STX11 protein of membrane fractions isolated from peripheral blood mononuclear cells confirms the different Stx11 genotypes. Membrane enriched fractions were separated by SDS-PAGE, blotted and probed with an antibody directed against the Nterminus of the STX11 protein. SNAP23 was used as a loading control. (D) Histochemistry of WT and Stx11-/- mice. Spleen, lymph node and thymus sections were stained with hematoxylin and eosin. Photos were taken with a light microscope under 100x magnification.

Figure S2. The cytotoxic impairment in Stx11-/- NK cells is not reversed after IL-2 stimulation. Specific lysis was assessed by release of cytoplasmic lactate dehydrogenase in vitro from YAC-1 cells co-incubated with WT or Stx11-/- LAK cells at various effector/target ratios (E:T). Results are expressed as mean ± SD. Representative results of one out of two independent experiments are shown.

Figure S3. Stx11-/- NK and CD8+ T cells display a phenotype comparable to WT counterparts. (A, B) Flow cytometry analysis of WT or Stx11-/- NK and CD8+ T cells freshly isolated from spleen. Plots are gated on SSC and NKp46+ (A) or SSC and CD8+ T cells (B). Numbers in plots indicate percent of positive cells. Shaded histograms show marker expression as indicated, bold lines show the corresponding isotype control. One representative experiment is shown (n=5-6 mice).

Table S1. Cell counts in primary and secondary lymphatic organs of the immune system.

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