JCS03459 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. (a) Six micrograms of Vn were resolved by 10% SDS-PAGE. The gel was stained as previously described (http://www.fhcrc.org/science/labs/hahn/methods/biochem_meth/silver _stain.html). (b) Purified human Vn was labeled with Alexa Fluor 568 and submitted to SDS-PAGE followed by UV illumination. Molecular sizes are indicated (kDa).

• Supplemental Figure S2 -

  Fig. S2. (a) Localization of ligand binding domains in Vn and PrP^C molecules. The interaction sites for plasminogen activator inhibitor-1 (PAI-1; 1-44; 348-370), urokinase receptor (uPAR;1-44), integrins (45-47), thrombin-anti-thrombin III complex (TAT;53-64), collagen (53-64), cellular prion protein (PrP^C;307-320), plaminogen (332-348) and heparin (348-361) were labeled in the Vn molecule. In PrP^C molecule, the binding sites for glycosaminoglycans (GAG; 23-35), Cu²⁺ ions (51-90), Vn (105-119), neurotrophin p75 receptor (p75; 106-126), stress-inducible protein 1 (STI1; 113-128), Ln (173-192), neural adhesion molecule (NCAM;144-154) and laminin receptor 37/67 kDa (144-179) were indicated (Schvartz et al., 1999; Coitinho et al., 2006; Lee et al., 2003). (b) Competition assay with Vn ligands. PrP^C was added to polystyrene wells and 0.5 μg ¹³¹I-Vn were pre-incubated with increasing amounts (pmol) of unlabeled Vn, Collagen I, Collagen IV, Heparin or BSA before the addition into the wells. The wells were washed and radioactivity levels measured. Percentage of binding different from control. *P<0.05 using Student’s t-test.

• Supplemental Figure S3 -

  Fig. S3. Evolutionary conservation of the PrP^C binding domain in Vn. (a) Cross-species sequence comparison of the PrP^C binding site in the Vn molecule. Amino acids presenting divergence are shown in grey. (b) Binding assay between PrP^C and Vn from human and chicken origin. PrP^C was added to polystyrene wells and incubated with ¹²⁵I-labeled human or chicken Vn. The wells were washed and radioactivity levels measured. (c) Competition assay with Vn peptides. PrP^C was added to polystyrene wells and pre-incubated with Vn peptides before addition of ¹²⁵I-Vn. The wells were washed and radioactivity levels measured. PrP^C-Vn binding (no peptide added) was set as 100%. *P<0.01 compared with total binding using Student’s t-test.

• Supplemental Figure S4 -

  Fig. S4. Vn binding to the surface of ZrchI Prnp^+/+ cells is equivalent to that from ZrchI Prnp^0/0 ones. Primary mouse embryonic fibroblasts (PMEF) were obtained from E12.5 ZrchI Prnp^0/0 and ZrchI Prnp^+/+ embryos by tissue trypsin digestion and mechanical dissociation. Cells were cultured for 48 hours in DMEM (Invitrogen) plus 10% FCS. Flow cytometry was performed as follows: cells were removed from the culture flask by trypsin treatment and membrane recovery was performed one hour at 37°C in DMEM 10%FCS. (a) Cells were incubated in PBS 0.5% BSA for 1 hour at 37°C in the absence (left panels) or presence (right panels) of 5 μg Alexa Fluor 568-Vn. After washing, fluorescence was measured in a FACScalibur (Becton, Dickinson and Company). (b) Alternatively cells were incubated with biotinylated primary antibody against αv integrin subunit (Becton, Dickinson and Company) followed by streptavidin-FITC. After washing, fluorescence was measured by FACScalibur. Graph shows mean fluorescence value from three independent experiments. *P<0.05 compared with ZrchI Prnp^+/+ cells using Student’s t-test.