Tat protein and its N-terminal fragment Tat 1–45 interact physically with TLR4-MD2 and MD2 but not with CD14. A) rh TLR4-MD2, MD2 and CD14 were coated at 1 μg/ml in the wells. After incubation with GST-Tat 1–101, GST-Tat 1–45, GST-Tat 30–72 or GST control (1 μM), interaction of Tat with coated rh proteins was analyzed by ELISA. The data represent OD at 450 nm and are representative of one of three independent experiments. B-C) GST-pull down experiments: GST-Tat 1–101, GST-Tat 1–45, GST-Tat 30–72 or GST control (1 μM) coupled to glutathione-agarose beads were incubated B) with recombinant TLR4-MD2, MD2 and CD14 (1 μg/ml) or C) with 500 μg of cellular extracts from HEK cell expressing TLR4-MD2-CD14 or not. After washes, retained and unretained proteins fractions were evaluated by SDS-PAGE and western blot using antibodies directed against CD14, MD2, TLR4 and GST. D) Evaluation of the capacity of recombinant TLR4 to interact with recombinant MD2. rh TLR4 was coated at 1 μg/ml in the wells. After incubation with various concentrations of MD2 (1-10 μg/ml). TLR4-MD2 interaction was analyzed by ELISA using anti-MD2 monoclonal antibodies. E) rhMD2 and rhTLR4-MD2 compete for Tat-rhMD2 interaction: GST-Tat 1–101 (0.1 μM) were pre-incubated for 1 h with PBS (control) or with increasing amounts of soluble rhMD2, rhTLR4-MD2, rhTLR4 or rhCD14 before incubation with the coated rhMD2. Binding of Tat to rhMD2 was analyzed as described above by measuring OD at 450 nm. The data represent OD at 450 nm and are representative of one of three independent experiments.