Fig. 5.

Effects of ciprofloxacin and levofloxacin on LPS binding and LPS-induced TLR4 dimerization. Ba/F3 cells expressing TLR4-Flag (TLR4-F), TLR4-GFP (TLR4-G), MD2-Flag, and CD14 were pretreated with 500 μg/ml ciprofloxacin (CPFX) or levofloxacin (LVFX) for 1 h and then stimulated with 250 ng/ml of a biotinylated LPS (bLPS) or c LPS for 30 min. Cell lysates were immunoprecipitated with mouse anti-GFP antibody. The immunoblotted proteins were exposed to a streptavidin-HRP (upper) or chicken anti-GFP antibody (lower), and the ratio of Biotin-LPS/TLR4-GFP is shown in (b); c mouse anti-Flag (upper) or chicken anti-GFP (lower) antibodies and the ratio of TLR4-Flag/TLR4-GFP is shown in (d). Experiments were performed five times and representative images are shown. Data, expressed as percentage of LPS-stimulated cells, are shown as means ± SEM (n = 5). Data were analyzed by Kruskal-Wallis test (p = 0.0021) followed by Dunn’s multiple comparison test. *p < 0.05 and **p < 0.01 versus LPS stimulation. bLPS biotinylated LPS, Bio biotin, IP immunoprecipitation, IB immunoblot, Veh vehicle