Figure 1.

Preparation of recombinant GrpE and analysis of primary characteristics of DC maturation by GrpE treatment. (Aa) SDS–PAGE analysis of purified GrpE by Ni-NTA (M, markers; Bands, GrpE loading amount). (Ab) Western blot analysis of purified GrpE (10 μg) using mouse anti-His Ab. (B) Gating strategy of purified CD11c⁺ DCs. (C) Cytokine production by GrpE-treated DCs. DCs were generated by stimulating magnetic bead-purified immature DCs with LPS (100 ng/mL), GrpE (5 or 10 μg/mL) for 24 h, followed by ELISA for IL-12p70, TNF-α, IL-6, IL-1β, and IL-10 production. (D) Analysis of TNF-α, IL-12p70, and IL-10 expression in DCs by intracellular cytokine staining after 12 h at 37°C in the presence of GolgiPlug (1 μg/mL). One representative plot from three independent experiments is shown. (E) CD11c⁺ DCs were stained with anti-CD80, CD86, and MHC class I and II mAb and the expression levels of surface molecules were measured. One representative plot from three independent experiments is shown. (F) GrpE and LPS were heated for 1 h at 100°C (left panels) or digested with PMB (middle panels) or PK (right panels) before adding it to DC culture, as described in the section Materials and Methods. After 24 h treatment, TNF-α and IL-6 levels in the supernatant of DCs were analyzed using ELISA. These results are expressed as the mean ± SD (n = 3 samples) of representative result in three experiments (^*p < 0.05, ^**p < 0.01, or ^***p < 0.001). (G) DCs treated with GrpE or LPS were harvested 24 h later. The DCs were stained with anti-CD11c, annexin V, and PI and analyzed by flow cytometry. One representative plot from three independent experiments is shown. Con denotes untreated DCs.