Torilin Inhibits Inflammation by Limiting TAK1-Mediated MAP Kinase and NF-κB Activation

Figure 6

Torilin suppresses LPS-induced IKK and MAP3K complex activation and inhibits the TAK1 interaction with the components of MAPK and NF-κB signaling pathways. RAW macrophage cells were incubated with torilin or vehicle for 30 min and then stimulated with LPS as indicated in the figures. (a) Lysates from torilin or vehicle treated-cells were immunoblotted to detect the activation status of IKK. (b) Immunoblots depicting the time dependent effects of torilin in TAK1, MKK4, and IKK phosphorylation. (c) The kinase activity of the TAK1 was determined after immunoprecipitates were resuspended in kinase buffer and assayed for kinase activity as described in Materials and Methods. (d) Cell lysates were immunoprecipitated by incubating overnight with anti-TAK1 antibody and then incubated with protein A-Sepharose (PAS) for 4 h at 4°C. Precipitated proteins were separated by SDS-PAGE and immunoblotted to detect P-TAK1, TAK1, p-MKK4, MKK4, p-IKKα/β, IKK, and β-actin. Equivalent protein loading was verified by reprobing for the respective total antibodies and β-actin. Images are representative of 4 or more independent experiments. Values in bar graphs are means ± SE of at least 4 separate experiments performed in triplicate. , versus control.

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