Figure 3

Effect of OMC on LPS-dependent activation of AP-1, NF-κB and STAT3 in RAW264.7 cells. (A) AP-1 and (B) NF-κB DNA-binding activity was determined by gel shift analysis in nuclear extracts isolated from RAW264.7 cells pretreated with OMC (0, 50 100 μg/ml) for 24 hours followed by the stimulation with LPS (1 μg/ml) for an additional 30 minutes. Nuclear extracts were subjected to EMSA with a [³²P]-labeled AP-1 or [³²P]-labeled NF-κB probe as described in Materials and Methods. The specificity of DNA-binding was confirmed by competitive gel shift with cold AP-1, NF-κB, or unrelated DNA (URL). (C) STAT3 activity was evaluated in whole cell extracts treated with OMC for 24 hours and LPS for 30 minutes by Western blot analysis with anti-phospho-STAT3 antibody. The equal protein loading was verified with anti-STAT3 antibody. The results are representative of three separate experiments.