Intracellular localization and multimer pattern of mutant p.Cys2811Ala. wtVWF (A) and VWF mutant p.Cys2811Ala (D) were transiently expressed in HEK293 cells. Forty-eight hours after transfection, cells were fixed and VWF proteins (A,D) and PDI (B,E) were detected by indirect immunofluorescence employing rabbit anti-VWF and mouse anti-PDI antibodies, respectively. Z-stacks were recorded with a confocal microscope using an HC PL APO CS2 63.0 × 1.40 oil ultraviolet objective and the following settings: image size of 512 × 512 pixels, laser power of the 543 and 488 lasers was set to 9% and 20%, respectively. Overlays are shown in (C) and (F). Three-dimensional reconstruction was performed using the LAS software (Leica). Scale bars represent 10 µm. For movies of the rotating complete 3-dimensional reconstruction, please refer to supplemental Videos 1 and 2. (G) Multimer analysis of recombinant wtVWF and mutant p.Cys2811Ala was performed by sodium dodecyl sulfate–agarose gel electrophoresis and immunoblotting onto a nitrocellulose membrane with luminescent visualization. The figure is composed of one gel. The black line indicates deleted lanes with mutants not relevant for this study. Additional bands in mutant p.Cys2811Ala resulting from odd-numbered multimers are indicated by black arrows. For resolution of the monomer and dimer band, please refer to supplemental Figure 6.