Sialyl Residues Modulate LPS-Mediated Signaling through the Toll-Like Receptor 4 Complex
Figure 4
Recombinant human TLR4-His/MD2-His proteins expressed in the mammalian NS0 cell line were separated by SDS-PAGE and analyzed on immunoblot for binding to lectin SNA, MAAII and to anti-His antibody (A). Recombinant human TLR4-His/MD2-His expressed in NS0 cells (lane 1) or recombinant human MD2-His expressed in E. coli (lane 2) were separated by SDS-PAGE and probed by lectin blot with SNA (left) and on immunoblot with anti-His antibody (right) (B). Recombinant human CD14 (lane 1) were separated by SDS-PAGE and probed by lectin blot with SNA (left) and MAAII (right). The highly sialylated glycoprotein, fetuin (lane 2) and asialofetuin (lane 3) were included as positive and negative controls respectively (C). HEK293T cells were transfected with control pcDNA, or plasmids encoding TLR4-YFP alone or with MD2 and CD14 expression plasmids, and proteins from cell lysates were immunoprecipitated with ant-GFP antibody and probed on immunoblot with either anti-GFP antibody (top) or SNA (bottom) (D). Proteins from the medium of MD2-transfected cells were immunoprecipitated with anti-FLAG antibody and probed on immunoblot with anti-FLAG antibody (left) or SNA (right) (E). The expected molecular weights of MD2 and TLR4 are indicated by arrows. Results shown are representative of data from at least 2 independent experiments, each with similar results.