Intracelluar localization of VWF N-linked glycan mutants. Five days after transfection, HEK293 cells were fixed and stained with polyclonal rabbit anti-VWF and goat anti–rabbit AlexaFluor 488 and visualized under a laser scanning confocal microscope LSM510 META. Images in panels Ai through iv are representative images of typical staining patterns observed for (i) wtVWF, (ii) N99Q, (iii) N857Q, and (iv) N2790Q. Images in panels Av through vii are representative of a small number of cells transfected with N99Q (v), N857Q (vi), and N2790Q (vii) that were able to form pseudo Webiel-Palade bodies. (B) Ten separate fields of view were taken for each variant from 4 separate experiments and the number of cells with at least 5 visible WPB counted and expressed as a percentage of the total number of cells expression WPB. (C) Five transfection HEK293 cells were stimulated with 100 ng/mL PMA and VWF concentration analyzed by VWF ELISA. ■: No PMA; □, plus PMA