Expression levels, activity, and site specificity of S119F. (A left) Representative Western blot of endogenous ADAMTS13 in acute (AC) and remission (RE) phase of the propositus and in NHP. Blotted protein was detected with anti-ADAMTS13 mAb17B10 and GAM-HRP. Secondary antibody al1 was negative (not shown). (A middle and right) Transient expression of wild-type rADAMTS13 (WT), and mutants S119F (SF) and S119F/Q448E (SFQE). Protein in medium (middle) and cell lysate (right) was detected with peroxidase-labeled anti-V5 antibodies. (B) VWF collagen binding ELISA to assess residual VWF activity. Purified multimeric VWF (10 μg/mL) was incubated with 0.6nM ADAMTS13 in NHP (●), or 0.6nM rADAMTS13 S119F (♦) or S119F/Q448E (▾, dotted line) for 16 hours in 1M urea. All reactions were inhibited by 10mM ethylenediaminetetraacetic acid (EDTA; dashed lines) as shown for NHP (○) and S119F (◇). (C) Analogous samples as in panel B, in the presence (+) or absence (−) of EDTA, were analyzed in Western blot under reducing conditions where VWF product bands were detected by anti-A1 domain mAbs and GAM-HRP.