N-linked glycosylation of the VWF CK domain. (A) Multimer analysis was performed on lysate samples from HEK293T cells transfected with wtVWF and VWF-N2790Q. Mutant N2790Q media was concentrated approximately 20-fold for multimer analysis. (B) Cell lysate samples were collected at chase times 0, 2, 4, 18, and 24 hours and after immunoprecipitation were analyzed by SDS-PAGE under nonreducing conditions. *: Pro-VWF, ■: mature VWF. (C) The VWF CK domain wild type and N2790Q were transiently expressed in HEK293T cells and conditioned media and cell lysate samples analyzed by SDS-PAGE under reducing and nonreducing conditions. CK domain was detected with mouse monoclonal anti-myc antibody followed by goat anti–mouse HRP. The difference in migration between wtCK and CK-N2790Q confirms the absence of the N-linked glycan chain.