FIG. 4.

Analysis of the nature and accessibility of the two oligosaccharides of extracellular NS1. (a) Extracellular NS1 was mock treated (mt) or treated for 1 h at 37°C with endo H (H), endo F (F), or NPGase F (N) in its monomeric form. Samples were placed in nonreducing Laemmli sample buffer and separated by SDS–12% PAGE, and the resulting products were detected by immunoblotting after transfer to nitrocellulose. “p” indicates the presence of a polymannose-type oligosaccharide; “c” indicates a complex-type oligosaccharide on the protein. NS1(p)₀ is the deglycosylated form of the protein. (b) Purified NS1 was either mock treated or treated with NPGase F for 1 h at 37°C in 10 mM sodium phosphate buffer, pH 7.5. One sample was further treated by endo H after the pH was readjusted to 5.5 with 50 mM citrate buffer. All samples were subjected to SDS–12% PAGE with or without preliminary heat denaturation in nonreducing Laemmli sample buffer.