Figure 5: Ang II induces MD2/TLR4 complex formation in renal cells.

Effects of MD2 blockade by (A) L6H21 or (B) siRNA-mediated knock-down on Ang II-induced MD2/TLR4 complex formation were determined. NRK-52E cells were pretreated with L6H21 (2.5, 5, 10 μM) for 1 h, or transfected with 1 μg MD2-specific siRNA (Methods), stimulated with Ang II (1 mM) for 30 min, and cell lysates were co-immunoprecipitated (IP) with anti-MD2 or -TLR4 antibodies, and Western blot analysis (IB) made to detect TLR4 or MyD88; n = 4 independent determinations. The gels were run under the same experimental conditions. Shown are cropped gels/blots (The gels/blots with indicated cropping lines are shown in Supplementary Figure S6). (C) The effects of L6H21 on Ang II binding to cell surface was determined. NRK-52E cells were incubated with Ang II conjugated with fluorescent FITC (FITC-Ang II; 50 mg/ml) with or without L6H21 (2.5, 5.0, 10 μM), and flow cytometry was used to detect cell-bound fluorescence; shown is a representative flow analysis with the median fluorescence intensity (MFI) indicated for each group; n = 4 independent determinations.