Fig. 3.

Complex formation of ZNP/ZBP and its delivery into DCs. a Relative affinity of 1× ZBP and 3× ZBP is assessed by measuring fluorescent intensity of ZNP-peptide complex. 50 μg of ZNPs were incubated with indicated amount of peptide and the fluorescent intensities of the complex were measured after washing with PBS. b Gel electrophoresis data showing the relative fraction of ZNP-bound (P, pellet) or unbound (S, supernatant) ScaA after incubation with ZNPs and ScaA with or without 3× ZBP fusion. Relative intensity of the protein bands (P and S) was indicated below. c Intracellular delivery of ZNP/ZBP complex into DCs was assessed by fluorescence confocal microscopy after incubation of DC2.4 cells with ZNP and 3× ZBP-FITC complex. Lysosomes (red) were stained with LysoTracker. d Intracellular delivery of ZNP/ZBP-ScaA complex into DCs was assessed by fluorescence confocal microscopy after incubation of DC2.4 cells with ZNP and 3xZBP-ScaA complex. ScaA antigens (green) and Lysosomes (red) were stained with anti-ScaA antibody and LysoTracker, respectively. Intracellular co-localization of ZNP/ZBP-ScaA complexes with lysosomes were assessed by confocal imaging of z-stacks and orthogonal views (yz and xz) were shown in left and bottom panels. DIC differential interference contrast. White bar 10 μm