FIG. 4.

Asn⁵²⁶ and Asn⁵⁷⁵ are glycosylated in the presence of MD-2. (a) After plating 293 cells in 6-cm-diameter dishes, they were transfected with plasmids (5 μg each) for the TLR4 mutant with or without MD-2. After 24 h, cellular extracts were prepared from the cells and the extracts (10 μg protein) were analyzed for TLR4 and MD-2 by Western blotting (WB) with a rabbit anti-EIAV (α-EIAV) antiserum. wt, wild type; hTLR4, human TLR4. (b) After plating 293 cells in 6-cm-diameter dishes, they were transfected with plasmids (5 μg each) for the TLR4 mutant and MD-2. After 24 h, the prepared cellular extracts (10 μg protein) were either untreated or treated with PNGase F. The extracts were then analyzed for TLR4 and MD-2 by Western blotting with the rabbit anti-EIAV antiserum.