Figure 4: PA interacts directly with MD2 to activate TLR4 signalling.

(a) Flow cytometric analysis showing the effects of MD2 blockade on the binding of FITC-PA (50 μM) to the cell surface of mouse macrophages. MD2 was blocked with either anti-MD2 (100 ng ml−1) or L6H9 (1 and 10 μM). (b,c) Representative graphs from surface plasmon resonance spectroscopy analysis showing the binding of increasing concentrations of PA with human recombinant MD2 (b) or TLR4 (c). The Ka and KD values are indicated on the upper left. (d) Binding of biotin-PA with rhMD2 pre-absorbed in the ELISA plate (OD values reported as mean±s.e.m. and analysed by Student’s t-test; *P<0.05, **P<0.01, compared with biotin-PA group). n=4 for all in vitro experiments.