Deletion of the CUB domains or a mutation in the second CUB domain of ADAMTS13 abolishes or reverses apical sorting. MDCK cells were stably transfected with full-length ADAMTS13 (FL), CUB domain–deleted ADAMTS13 (delCUB), and a mutant ADAMTS13 (4143-4144insA) that deletes the second CUB domain and appends amino acid REQPG after S1381 as shown in panel A. The cells were grown on the Transwell filter units, and the conditioned media were collected from apical and basolateral domains. ADAMTS13 and mutants secreted were determined in the equal volume of the concentrated conditioned media by Western blotting with anti–V5 IgG (panel B). The relative amount of protein secreted into the apical domain of MDCK cells was quantified by densitometry of the signals on X-ray film with ImageJ software and is shown in panel C. The entries here represent the means ± SD from 3 independent experiments. M indicates metalloprotease domain; Dis, disintegrin domain; T1 to T8, first to eighth thrombospondin type 1 repeat; CysR, cysteine-rich domain; Spa, spacer domain; and CUB, complement C1r/C1s, urinary EGF, and bone morphogenetic protein domain.