Figure 10

Stimulation of hTLR4/MD-2 by ApoA-1 and Hsp70 produced in endotoxin-free E. coli strains. The proteins were minimally purified by IMAC and assayed with HEK-Blue hTLR4 cells for their ability to activate NF-κB-dependent SEAP expression (A and C). HEK-Blue Null2 cells served as a control (B and D). Relative NF-κB induction was measured following stimulation of HEK-Blue hTLR4 and Null2 cells with tenfold serial dilutions of ApoA-1 (A and B) and Hsp70 (C and D) samples obtained by heterologous expression in BW30270/pApo404, KPM318/pApo404 and KPM335/pApo404, and BL21 (DE3)/pHsp70His and KPM404/pHsp70His, respectively. The values represent the means and standard deviations from three individual experiments. In all experiments, the ApoA-1 and Hsp70 samples did not activate NF-κB-dependent SEAP expression in HEK-Blue Null2 cells, suggesting that NF-κB-dependent SEAP expression was due to specific activation of the hTLR4/MD-2 signaling pathway in HEK-Blue hTLR4 cells.