Phenotypic analysis of Sec22b^−/− mice. (A) Western Blotting of Sec22b expression in peritoneal macrophages and CD11c⁺ splenic DCs (top: purified by negative (neg. sel.) and positive (pos. sel.) selection for CD11c⁺ cells), as well as B and T cells (bottom), isolated from Sec22b^+/+ and Sec22b^−/− mice. Shown is one representative experiment of three independent experiments. (B) Phenotypic characterization of DC subsets in spleens and lungs from Sec22b^−/− and Sec22b^+/+ mice. Gating strategy are shown for splenic DCs (left), percentages of CD8⁺ and CD11b⁺ DCs subpopulations in spleen (middle), and cell numbers (right). Data shown are means of four independent biological replicates. DC populations were analyzed by gating on CD11c^high, MHC class II^high/mid and CD8/CD11b. (C) Gating strategy are shown for lung DCs (left), percentages of CD103⁺ and CD11b⁺ DCs subpopulations in spleen (middle), and cell counting (right). Data shown are means of four independent biological replicates and each value. DC populations were analyzed by gating on CD11c^high, MHC class II^high, then in Siglec F^low (to discriminate from alveolar macrophages), and finally in CD103/CD11b. (D) Phenotypic analysis of BMDCs. Western blotting analysis of Sec22b expression in BMDCs generated from Sec22b^+/+ and Sec22b^−/− mice. (E) Shown are percentages of CD11c^highCD11b^high cells (left) and expression of the costimulatory molecules CD40, CD80, CD86, and MHC class II upon TLR4 engagement with LPS (right). (F) Expression of MHC class I H2-K^b and D^b. For all of the BMDCs results, shown are the pooled data from at least three independent experiments (with the exception of LPS histograms showing one representative experiment of four independent experiments). All results in this figure were analyzed by two-way ANOVA with Bonferroni’s multiple comparisons test.