Figure 7: MST measurements of the interaction between CnB and the TLR4 receptor complexes.

(a) Measurement of rhCnB binding to the purified TLR4 ectodomain. (b) Measurement of CnB binding to the full-length TLR4 from transfected HEK293 lysates. (c) Measurement of rhCnB binding to purified soluble CD14. (d) Measurement of rhCnB binding to full-length membrane-anchored CD14 from transfected HEK293 lysates. (e) Measurement of the binding of rhCnB to secreted MD2 in the supernatant from transfected HEK293 cells. (f) Measurement of rhCnB binding to MD2 in the transfected Hek293 cell lysates. The purified TLR4 ectodomain or soluble CD14 was labelled with DyLight 488, and the concentration of labelled protein was adjusted to 20 nM. GFP-tagged TLR4, CD14 and MD2 constructs were transfected into HEK293 cells, incubated for 48 h, and lysed with RIPA buffer. Secreted MD2 was obtained from the supernatant of the MD2-transfected hek293 cells. The lysates were diluted according to fluorescence intensity. The recombinant CnB protein was dissolved to a 500 μM concentration using MST buffer and 16 1:1 dilution samples were prepared. The labelled proteins or GFP-tagged receptors lysates were added into each ligand dilution and mixed. After 10 min incubation, each solution was added to Standard Treated Capillaries for Thermophoresis. The data were analysed using NT. Analysis software. All data are representative of at least two independent experiments. (g) Model depicting the recognition of exogenous CnB. Membrane-anchored or soluble CD14 first recognized CnB and transported it to the TLR4/MD2 complex located on the plasma membrane, followed by internalization of the CnB/TLR4 receptor complexes and signalling through TLR4. Free MD2 also recognized and bound to CnB, although the affinity was lower than that of the MD2 on the plasma membrane.