FIGURE 5.

Both the MAM domain and the adjacent linker region between MAM and F5/8 domain are necessary and sufficient for polysialylation. The Fc-linked NRP MAM and LM chimeras were coexpressed with ST8SiaIV in COS-1 cells. The proteins were precipitated (IP) from the cell medium using protein A-Sepharose beads. Polysialylation of these proteins was assessed by immunoblotting (IB) using the anti-polySia 12F8 antibody (top). Relative protein expression levels were determined by removing the bound protein from half of the protein A-Sepharose beads by boiling and immunoblotting with an HRP-linked anti-human IgG (bottom). Quantification of the experimental results was performed as described under “Experimental Procedures” with data from four different experiments with error bars representing S.D. Statistical analysis was performed using unpaired Student's t tests. *, 0.01 < p < 0.05; ***, 0.0001 < p < 0.001; ****, p ≤ 0.0001; ns, p ≥ 0.05 with respect to wild type NRP-2, which is normalized to 100%. Other comparisons are indicated by a line above the compared bars in the graph.