Fig. 2. Cleavage of GST-VWF73-H by ADAMTS13 and the carboxyl-terminal truncated mutants.
Purified GST-VWF73-H (200 ng) was incubated with 16 nM ADAMTS13 and the various carboxyl-terminal deleted mutants of ADAMTS13 in the absence (−) or presence (+) of 10 mM EDTA for 3 h (A) and 24 h (B), respectively, in a total volume of 40 μl. Western blotting with rabbit anti-GST IgG and chemiluminescent reagents detected the cleavage of GST-VWF73-H. Construct derivations are as in Fig. 1. The arrows indicate intact GST-VWF73-H (38.4 kDa), and the arrowheads indicate amino-terminal fragments of GST-VWF73-H (34.4 kDa) generated by cleavage of the Tyr–Met bond. The double stars (30 kDa) indicate amino-terminal fragments generated by cleavage of GST-VWF73-H at the site other than the Tyr–Met bond.