Fig. 1. LASAG specifically acts via inhibition of IKK-mediated NF-κB activation and has no impact on virus-induced MAPK activation.

a Activation of the NF-κB signaling pathway in A549 cells via TNF-α leads to degradation of IκB. IκB degradation and a NF-κB activation are inhibited after treatment of A549 cells with either 10 mM LASAG (BAY 81–8781) or 10 mM ASA. ERK2 represents the loading control. b, c, d LASAG inhibits IKK-mediated transcriptional activation of NF-κB-dependent promoters. A549 cells were transfected with plasmids carrying a NF-κB-specific promoter element in front of a luciferase gene b or the promoter constructs of NF-κB-dependent genes IL-6 C and IL-8 d. Cells were co-transfected with either empty vector or a plasmid expressing a wt form of IKK2 that is active upon overexpression. At 16 h post-transfection, cells were treated with solvent or 5 mM LASAG for an additional 6 h. Cells were then lysed and promoter activity was determined by measuring luciferase activity. The results show the mean of three independent experiments. P < 0.05 = *; P < 0.01 = **; P < 0.005 = ***. e LASAG does not have non-specific effects on virus-induced activity of mitogen-activated protein kinases (MAPK) JNK and p38. A549 lung epithelial cells were either left uninfected (lanes 1–3) or were infected with IAV A/FPV/Bratislava/79 (H7N7) (MOI = 5) for 4 or 8 h, respectively (lanes 4–9). Infected cells were either left untreated (lane 1–5) or treated with either 5 mM (lanes 6 and 7) or 7 mM LASAG (lanes 8 and 9) immediately after infection. Cells were then lysed, and protein lysates were separated by PAGE and blotted onto nitrocellulose membranes. Membranes were then incubated with antibodies against phosphorylated active forms of MAPKs JNK and p38. Pan-JNK1 and p38 blots served as loading controls