N-glycosylation affects the secretion of ADAMTS13. (A) Tunicamycin suppresses the secretion of ADAMTS13. HEK 293T cells were transfected in the absence (B) or presence (T) of tunicamycin. Cell lysates (C) and concentrated culture media (M) were collected 72 hours after transfection and analyzed at equal volumes by SDS-PAGE and immunoblotting with anti-V5. The numbers to the left of the gels indicate the position of molecular weight markers in kilodaltons. (B) The effect of N to Q substitution on the secretion of ADAMTS13. Transfection of wild-type ADAMTS13 (WT) cDNA or N-glycosylation site mutants was performed in the absence of tunicamycin. Each percent ADAMTS13 secretion was determined from the concentration of ADAMTS13 in the culture medium, divided by the sum of protease concentration in the culture medium and cell lysate. The values were normalized against 100% for the mean of the WT ADAMTS13 secretion. Mean and SD values shown on the y-axis were based on at least 5 batches of transfections. *P < .01 by ANOVA with Dunnett postanalysis against the WT ADAMTS13.