Effect of N-linked glycosylation inhibitors on VWF expression and glycosylation of the VWFpp. (A) Domain diagram of von Willebrand Factor (VWF) showing the location of the 16 N-linked glycosylation sites, 4 in the propeptide and 12 in each mature subunit. (B) Wild-type (wt) VWF was transiently expressed in HEK293T in the presence of 1 μg/mL tunicamycin (TM), 100 μg/mL castanospermine (CST), or 10 μg/mL swainsonine (SW) and VWF levels in the cell media (■) or lysate (□) analyzed by ELISA 3 days after transfection. Error bars represent mean ± SEM of 3 separate experiments each performed in duplicate. (C) The multimer composition of VWF expressed with glycosylation inhibitors was determined by electrophoresis in 1.4% agarose gels followed by Western blotting. (D) Microtitre plates coated with anti–VWF-propeptide CLB-Pro35 were used to capture plasma (■) or recombinant (□) VWFpp and the glycan content assessed using lectins Concanavalin A (Con A), Elderberry bark lectin (EBL), and Ulex europeaus (Ulex. E). Anti–VWF-propeptide-HRP (CLB-Pro 14.3) was used to confirm capture of the propeptide and an anti–VWF-CK domain antibody was used to confirm the absence of mature VWF.