Generation of human anti-hDEC205 monoclonal antibodies. (A) Expression of soluble human DEC205-human IgG1 Fc (V5.hDEC205/hIgG1Fc) fusion protein. Left: CHO cells, transfected with control plasmid (lane 1) and V5.hDEC205/hIgG1Fc (lane 2), were lysed and analyzed by Western blotting with anti-V5 antibody. Right: purified human IgG1 Fc alone (lane 1) or V5.hDEC205/hIgG1Fc fusion proteins (lane 2) produced by transfected CHO cells were analyzed on SDS-PAGE and stained by Coomassie blue. (B) FACS labeling of CHO/hDEC205 and CHO/mDEC205 cells and (C) human MoDCs stained at 1 μg/mL with human anti-hDEC205 mAbs, as well as the previously described NLDC145 rat anti-mDEC205 and MG38.2 mouse anti-hDEC205. The MoDCs were treated with/without LPS (100 ng/mL) to generate mature and immature DCs. (D) Lysates from CHO/Neo (lane 1), CHO/hDEC205 (lane 2), and CHO/mDEC205 (lane 3) cells were analyzed by Western blotting with mAbs 3G9 anti-hDEC205, NLDC145 anti-mDEC205, and anti-actin. (E) A series of deletion constructs in the extracellular domain of human DEC205 were generated (left, underlines), each with a FLAG tag, and each FLAG-tagged deletion construct was expressed in 293T cells. To map the 3G9 epitope on DEC205, each cell lysate was analyzed by Western blotting with mAbs 3G9 and L5 anti-FLAG.