Regulation of NCTD on LPS induced NF-κB activation.

<p>A. Little influence was observed by NCTD on IκBα and MAPK signaling pathway. RAW264.7 cells were treated with PBS (containing 0.1% DMSO) or 1–10 μM NCTD for 24 h and then stimulated with 100 ng/ml LPS for different time. The protein levels of IκBα and MAPK signaling pathway in RAW264.7 cells were determined using western blotting. B. The translocation of p65 to nucleus was increased in NCTD treated macrophages. Cells (2×10⁶ cells/ml) were incubated with PBS (containing 0.1% DMSO) or 1–10 μM NCTD for 24 h and treated with 100 ng/ml LPS for 15 min. Cytoplasmic extracts (CE) and nuclear extract (NE) were prepared and analyzed by western blot analysis using specific antibodies. And the translocation of p65 to nuclear was also quantitatively analyzed. C. LPS induced DNA binding ability of NF-κB was enhanced by NCTD in a dose dependent manner. Cells were treated with PBS (containing 0.1% DMSO) or 1–10 μM NCTD at 37°C for 24 h and then activated with 100 ng/ml LPS for 30 min. Finally, nuclear extracts were prepared and then assayed for NF-κB activation by EMSA as described under Materials and Methods. Three independent experiments were performed. D, Diagram of NCTD facilitates LPS-mediated immune responses by up-regulation of AKT/NF-κB associated signaling pathway.