Expression of VWF in HA-HRH1 HEK293T. (A) Representative staining of transiently transfected cells with or without fixation and permeabilization. Fixed cells were costained for DNA (4′,6 diamidino-2-phenylindole) and VWF (Alexa-Fluor 488); a large number of Weibel-Palade body-like structures can be observed throughout the cytoplasm of both WT and all 4 mutant VWF-transfected cells. (The data are representative of 3 independent experiments in which each construct was transfected in 3 × 8-well slides. A minimum of 10 microscopic fields per well were photographed for a total of >240 fields analyzed per construct.) Live histamine-treated cells were costained with the plasma membrane marker CellMask DeepRed (red) and anti-VWF antibody (Alexa-Fluor 488). VWF-rich domains can be seen on the surface of some of the cells (magnified fields are shown in the right column). No differences were observed between WT and mutant VWF-expressing cells. (The data are representative of 3 independent experiments in which each construct was transfected in 3 × 8-well slides. A minimum of 5 microscopic fields per well were photographed for a total of >120 fields analyzed per construct.) Wide field images were captured at an original magnification of ×63 (1.4 aperture) and high-magnification images at ×100 (1.4 aperture). All images were captured on a LSM 510 Meta confocal microscope. Capture of live samples was performed at 37°C; all other samples were photographed at room temperature. Fixed samples were mounted in Vectashield mounting medium (Vector Laboratories, Burlingame, CA). All images were captured and processed with the LSM 510 software. (B) Samples taken from cells transfected with VWF WT, VWF/p.S1494C, VWF/p.S1534C, and VWF/p.S1494-p.S1534C were run on a 2% agarose 0.1% SDS gel. After transfer, membranes were analyzed for VWF expression. The dashed line indicates that part of the immunoblot is cropped to exclude irrelevant samples. All constructs expressed as multimerized proteins when expressed in HEK293T cells (n = 2).