Multimer distribution and intracellular localization of recombinant N528S-VWF. (A) Mutant N528S-VWF (lane 1) alone and coexpressed with wt VWF (lane 2) in 293 EBNA cells in the conditioned medium was assessed by SDS-agarose electrophoresis and Western blotting. Vertical line(s) have been inserted to indicate a repositioned gel lane. Recombinant wt VWF multimers are shown for comparison (lane 3). N528S-VWF multimers lack HMWM completely, whereas N528S coexpressed with wt VWF could not be distinguished from wt VWF alone. (B) AtT-20 cells were transiently transfected with (top panel) wild-type VWF or (bottom panel) N528S-VWF. Cells were immunostained for VWF and examined by confocal microscopy. N528S-VWF was not trafficked to storage granules.