Maturation and processing of HJV. (A) Schematic representation of HJV functional domains and localization of the studied mutations. SP indicates signal peptide; RGD, arginine-glycine–aspartic acid integrin-binding domain; ⋄, Cys residue; and •, Asn residue. The dotted line indicates the autoproteolytic site. The double line shows the peptide chosen for antibody production. (B) Characterization of the mouse polyclonal anti-HJV. Hela cells were transfected with empty vector (mock) or WT HJV-expressing construct (WT). HJV was synthesized by in vitro transcription and translation (TNT). *Unspecific band. Anti-HJV recognizes a major band, which corresponds to the mature HJV, and a faint band derived from the autoproteolysis of the VWF type D domain. (C-D) WT and mutants HJV (30 μg of total lysates) were treated (+) with Endo H (C) or PNGase F (D) following manufacturer's instructions. (E) The nonsense R326X mutation produces a truncated HJV that is partially glycosylated, as shown by the band shift after Endo H and PNGase F treatment. The arrows show the glycosylation state. Scales refer to relative molecular mass in kilodaltons.