Further analysis of the expression of VWF N-linked glycan mutants. (A) Conditioned media and cell lysate samples were collected after 0, 6, 18, and 24 hours chase and immunoprecipitated with Dynabeads protein G and polyclonal anti-VWF antibodies. Immunoprecipitates were analyzed by SDS-PAGE in 4% to 12% gels under reducing conditions and autoradiography. *: Pro-VWF; ■: mature VWF. (B) Glycosylation at N99, N857, N2400, and N2790 was blocked by mutating the third amino acid in the N-linked glycosylation consensus sequence. Mutants were expressed in HEK293T cells and expression analyzed by VWF ELISA. Error bars represent mean ± SD of 3 separate experiments each performed in duplicate. (C-D) The multimeric structure of wtVWF, N99Q, N857Q, N2400Q, and N2790Q in both the expressed media (C) and cell lysate (D) was assessed in 1.4% agarose gels and visualized with HRP-conjugated anti-VWF polyclonal antibodies.