Characterization of Zebrafish von Willebrand Factor Reveals Conservation of Domain Structure, Multimerization, and Intracellular Storage

Figure 2

Multimerization of zebrafish Vwf in mammalian cell culture demonstrates high molecular weight multimers similar to human VWF. HEK293T cells were transfected with pzVwf/V5-HISA, expressing V5-HIS tagged zebrafish Vwf (zVwf), or pCineoVWF, expressing untagged human VWF (hVWF). Normal human plasma (NHP) and zebrafish and human supernatants were separated by agarose gel electrophoresis, transferred by western blotting, and detected with either a pool of monoclonal anti-hVWF antibodies (Avw1, 5, 15, left panel) or a mixture of anti-V5 and anti-HIS antibodies (for tagged zVwf, right panel). The anti-V5/HIS combination detects zVwf with a multimer pattern, including high molecular weight multimers, indistinguishable from that typically observed for human VWF (brackets indicate high molecular weight multimers for both zebrafish and human VWF).