Figure 8.

Effects of SIF treatment on LPS-induced NF-κB nuclear translocation via the IKK/IκB signaling pathway. (A) Confocal microscope images of RAW264.7 cells stained with anti-NF-κB-p65 antibody (Alexa 488, green) and nuclei (DAPI, blue). RAW264.7 cells were pre-treated with SIF (1 mg/mL) for 12 h prior to LPS treatment for 15 min. Microscopic images of p65, nuclei and merged signals (Alexa 488 and DAPI) are shown in the upper, middle, and lower panels, respectively. All images were taken using an LSM-780 microscope (Carl Zeiss). Original magnification: ×63 with ×3 digital zoom, oil objective. Scale bar for the images indicates 5 µm. (B) IKK phosphorylation was detected using an immunoblotting assay with anti-phospho-IKKα/β, total-IKKα and IKKβ monoclonal antibodies, respectively. (C) Phosphorylation of p65 was detected using phospho-NF-κB-p65 and NF-κB-p65 antibodies. (D) IκB degradation was detected using an anti-IκBα monoclonal antibody. RAW264.7 cells were pre-treated with SIF at different concentrations (0–1 mg/mL) for 12 h. Total cell lysates were collected after 15 min LPS (0.3 µg/mL) treatment. GAPDH was used as a loading control. (E) Densitometric quantification of IKK phosphorylation, phosphorylation of p65 and IκB degradation were shown in the left, middle and right panels, respectively. The graph calculated by averaging the results were normalized each internal control from three independent experiments. Data represent the mean ± S.D (ratio to Con, significant compared with LPS alone, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).