728196.fig.003a
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728196.fig.003b
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Figure 3: Effects of Zuonin B on NF-κB activation and MAPK expression. (a) RAW264.7 cells were pretreated with different concentrations of Zuonin B for 1 h and stimulated with LPS (1 μg/mL) for another 1 h. Equal amounts of protein in the cell lysates were electrophoresed, and the levels of NF-κB (p65) protein in the cytosol and nucleus determined using specific antibodies for p65 and IκB-α. The respective protein levels of β-actin and PARP were used to confirm equal amounts of protein for electrophoresis. (b) Immunofluorescence staining for NF-κB p65. RAW264.7 cells were pretreated with 30 μM Zuonin B for 1 h, followed by stimulation with LPS for another 1 h. Cells were fixed, permeabilized, and incubated with a specific antibody for NF-κB p65, followed by Texas-red conjugated anti-rabbit Ig (red). The nuclei of the corresponding cells were visualized by DAPI staining (magnification 400). (c) Zuonin B inhibited LPS-stimulated phosphorylation of ERK1/2 and JNK in Raw264.7 cells. Cells were pretreated with different concentrations of Zuonin B for 1 h, and stimulated with LPS (1 μg/mL) for another 30 min. NC: untreated control cells; LPS: LPS only treatment.