LCLs retain DEC-205–targeted antigen longer than DCs. (A) LCLs, mDCs, or iDCs were incubated with 1 μg/mL control Ig-EBNA1 or αDEC-205-EBNA1 on ice for 30 minutes. Cells were then washed and incubated at 37°C for the indicated time periods. As a control for internalization, cells were left on ice or incubated with 10μM cytochalasin B (cyto B) in addition to αDEC-205-EBNA1 pulsing. Cells were then stained with PE-conjugated anti-mouse antibody. Data are mean values plus SD from 3 independent experiments and relative internalization is shown as percent maximum. (B) LCLs, mDCs, or iDCs were incubated without (−ve) or with 4 μg/mL αDEC-205-LMP1 on ice for 30 minutes. Cells were then washed and incubated at 37°C or left on ice as a control. Cell samples were lysed at the indicated time points and frozen. Protein lysates were separated by SDS/PAGE, transferred to PVDF membranes for Western blotting, and probed with a LMP1-specific mAb. Blots were also probed for actin as a loading control. One representative blot of 3 experiments is shown; mean values plus SD of relative protein content and percentage maximum from 3 independent experiments are also shown.