Abnormal trafficking of VWF in Vps33b^fl/fl-ER^T2 mice. (A) VWF levels were measured in MKs by immunoblot (left panel) and densitometry (right panel) using a rabbit polyclonal antibody anti-VWF (H-300) (1:1000), and a goat anti-rabbit and a goat anti-mouse horseradish peroxidase-conjugate secondary antibody (1:1000). β-Actin was used as a loading control (n = 3 mice per genotype). (B) Immunogold labeling (IEM) using the Tokuyasu method for double labeling of VWF and CD63 (i-iv) in MKs from Vps33b^fl/fl and Vps33b^fl/fl-ER^T2 mice. Black arrowheads, VWF 10 nm gold particles; black arrows, CD63 15 nm gold particles. Scale bar, 250 nm. A total of 15 MKs were imaged per genotype, 4 to 5 fields of view (4.98 × 3.32 μm) per MK taken at a magnification of ×30 000. (C) Fibrinogen uptake in cultured MKs after incubation with 488-fibrinogen for 2 hours. Mean ± SEM. A, atypical MVB II, MFI, mean fluorescence intensity; ns, not significant.