Effect of N-linked glycosylation mutation on the expression and secretion of VWF. VWF N-linked glycan mutants were expressed in HEK293T cells and VWF concentrations in the secreted media (A) and cell lysate (B) were analyzed by VWF ELISA. Error bars represent mean ± SEM of 3 separate experiments each performed in duplicate. Inset: Cell lysate samples of wtVWF and the VWFpp glycosylation mutants were analyzed by reducing SDS-PAGE in 3% to 8% gels followed by Western blotting with polyclonal anti–VWF-HRP