ADAMTS13 uptake by iDCs. (A) ADAMTS13-488 (50nM) was incubated with iDCs for 45 minutes at 37°C and cells were analyzed by FACS. The gray histogram represents control cells without ADAMTS13-488. (B) Cell-surface–bound versus internalized ADAMTS13 was determined by incubating iDCs with ADAMTS13-488 at 4°C or 37°C for 45 minutes. (C) Uptake was performed with increasing concentrations (0-200nM) of ADAMTS13-488 (45 minutes) or at prolonged time intervals (0-50 minutes with 50nM ADAMTS13-488). Graphs represent data from 2-3 independent experiments ± SD. Data are expressed as the percentage of mean fluorescence intensity at 37°C, where 100% corresponds to the highest mean fluorescence signal observed for individual experiments such as 50nM (B) or 200nM ADAMTS13-488 and 50 minutes of ADAMTS13-488 uptake (C). (D) ADAMTS13-488 was added to iDCs for 45 minutes at 37°C. DCs were fixed, permeabilized, and stained with anti-EEA1 Ab, followed by Alexa Fluor 568–labeled secondary Ab (EEA1 staining is shown in red).