Fig. 6. Biochemical characterization of Cvl2 proteins. (A) SDS-PAGE gel showing purified Cvl2-N (lanes 2, 5), Cvl2-CM (lanes 3, 6) and Cvl2-WT (lanes 4, 7) proteins expressed in SF9 cells under reducing (lanes 2-4) and non-reducing (lanes 5-7) conditions; bands in lane 4 correspond to the uncleaved protein (NC), and the N- and C-terminal cleavage products. (B) Biacore sensograms showing the binding of 50 (a), 100 (b), 300 (c) nM Cvl2-N to immobilized Bmp2. (C) Sensograms showing binding of 8 (a), 25 (b) and 75 (c) nM Chordin to immobilized Bmp2. (D) Binding of Chordin to Cvl2-N saturated immobilized Bmp2. At time zero, perfusion with 500 nM Cvl2-N was initiated. The saturation binding of Cvl2-N was set as zero. After 120 seconds, perfusion was continued with 500 nM Cvl2-N plus Chordin. In different cycles, 25 (b) and 75 (d) nM Chordin were applied. In between, sensograms were recorded in the absence of Chordin (a,c). Perfusion with buffer started at 240 seconds. The subtle shift upon perfusion with highly concentrated Chordin (b,d) might result from a slow, equilibrium-driven replacement of Cvl2-N by Chordin. (E-H) Western blots with anti-Myc antibody. (E) Differential distribution of Cvl2 proteins in HEK 293 cell cultures. Cells were transfected with empty vector (lanes 1,2), or plasmids encoding Cvl2-WT (lanes 3-5) or Cvl2-N (lanes 6,7). Cleaved/associated Cvl2-WT is characterized by a high molecular weight band in the absence of ME (lane 3), and a low molecular weight band in the presence of ME (lane 4). It is found only in the supernatant, whereas uncleaved Cvl2-wt (high molecular weight band in presence of ME) is found only in the ECM fraction (lanes 4,5). Cvl2-N is present only in the supernatant (lanes 6,7). (F) Elution profiles of Cvl2 proteins from heparin-coated sepharose beads. Cvl2-CM, Cvl2-CM393-396 and Cvl2-N display decreasing affinity to heparin. (G) Removal of a putative heparin binding site (amino acids 393-396) from Cvl2-CM leads to accumulation of the protein in the supernatant of HEK 293 cell cultures. (H) In injected zebrafish embryos, Cvl2-CM and Cvl2-CM393-396 are present in similar amounts, although the dorsalizing effect of Cvl2-CM393-396 is much weaker (compare with Table 1).