scFvNLDC-145targets tumor antigen into DCs in vivo. A schematic representation of expression vectors. scFvNLDC-145-HER2, scFvNLDC-145-neu, pcDNA3.1-HER2, or pcDNA3.1-neu encode under the control of a CMV promoter, all the fusion proteins consisting of an signal peptide, amino acid residues 22 to 561 of human HER2 or amino acid residues 22 to 582 of rat neu, and COOH-terminal His tag. The control plasmids pcDNA3.1-HER2 and pcDNA3.1-neu lack the NLDC-145 domains. B 293T cells grown in 100-mm dishes were transfected with various expression vectors using Lipofectamine 2000 (invitrogen). Immunoblot analysis of supernatants from 293T cells transfected with scFvNLDC-145-HER2, scFvNLDC-145-neu, pcDNA3.1-HER2 and pcDNA3.1-neu (lane 1, 2, 3 and 4). Vaccine proteins were probed with mouse anti-His tag mAb followed by HRP-conjugated secondary anti-mouse antibody. C left, scFvNLDC-145-EGFP plasmid and controls as indicated were injected i.m. into mice, spleens were removed 48, 60, and 72 h thereafter, and stained with PE-conjugated anti-CD11c antibody. The GFP fluorescence in splenocytes was measured by flow cytometry. Representative results from one animal of each group 60 h post-injection. right, the expression of EGFP (MW,~25 kDa) and scFvNLDC-145-EGFP (MW,~55 kDa) fusion protein in injected muscle tissues detected by western blotting using anti- EGFP antibody 24 h after plasmid injection with GAPDH (MW,42 kDa) as loading control. Lane 1, mice treated with pcDNA3.1 control vaccine; lane 2, mice treated with EGFP-encoding vaccine; lane 3, mice treated with scFvNLDC-145-EGFP vaccine; D percentages of GFP+ DC in total DC (left panel); Percentages of GFP+ in CD11c-negative splenocytes (right panel). Bars, SE. *, P < 0.01, scFvNLDC-145-EGFP compared with other groups.