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Blood, Vol. 107, Issue 10, 3922-3924, May 15, 2006
P-selectin binds to the D'-D3 domains of von Willebrand factor in Weibel-Palade bodies
Blood Michaux et al.
107: 3922
Supplemental materials for: Michaux et al
Files in this Data Supplement:
- Figure S1. VWF D’-D3 domains can bind P-selectin: controls (JPG, 23.6 KB)
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A) P-selectin is required to immunoprecipitate VWF.
HEK293 cells were nucleofected with the P-selectin luminal domain and either VWF-GFP or D1-D3-GFP (constructs are detailed in figures 1&2). Cells were lysed and P-selectin immunoprecipitated from the lysate by the monoclonal anti-P-selectin antibody AC1.2. The specificity of coimmunoprecipitation of VWF with P-selectin was determined by Western blot using a rabbit polyclonal anti-GFP antibody: When P-selectin is absent, (lane 5 and 7) VWF is not detected.
B) The pro-peptide does not bind P-selectin.
HEK293 cells were nucleofected with the P-selectin luminal domain and either full-length VWF, the pro-peptide or mature VWF (Fig 1A). Cells were lysed and P-selectin immunoprecipitated from the lysate by the monoclonal anti-P-selectin antibody AC1.2. VWF was detected by Western blot using the monoclonal anti-Pro antibody 239.1 (Haberichter et al, 2000; Blood, 95, 1808): although the pro-peptide is clearly visible in lane 1 and 2, it is absent after the co-IP (lane 5 and 6); lane 4 is empty. The very weak band visible in lane 5 (arrowhead) is probably due to a complex formed by the pro-peptide together with mature VWF and P-selectin. The specificity of the anti-Pro antibody was tested in lane 3 were it fails to detect anything after expression of mature VWF only. Note that there is a small difference in size between the pro-peptide detected after cleavage (lane 1) and after expression of Pro on its own. See Fig S2 for details.
- Figure S2. The variable s8ize of the pro-peptide (JPG, 13.1 KB)
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HEK293 cells were nucleofected with either FL-VWF (lane 1, 3 and 6), mature VWF (lane 5 and 8), or the pro-peptide on its own (lane 4 and 7). Cells were lysed and VWF was detected by Western blot using a rabbit polyclonal anti-VWF antibody (lane 1-2) or two different monoclonal anti-Pro antibodies (landes 3-5: 242.6; lanes 6-8: 242.5; Haberichter et al 2005, Blood 105; 145), both distinct from the one used in Fig S1. In all cases, the pro-peptide, when expressed on its own, is detected as slightly smaller than when generated by cleavage from mature VWF after expression of FL-VWF. Since the Pro contstruct is designed to generate a protein of exactly the size of the cleaved pro-peptide (Haberichter et al, 2000), we propose that post-translational modifications of the pro-peptide differ depending on the context.
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