Figure 5

Analysis of Sp1 response element in CPT1A promoter region. (A) Electrophoretic mobility shift assay (EMSA) was performed for identification of Sp1 binding to the CPT1A promoter region from –50 to –5 in vitro. DNA-protein complexes were formed upon addition of nuclear extracts from vehicle or GBE-treated HepG2s (lane 2, 3). The binding specificity was competed by excess (×100) unlabeled (cold) probes (lane 4). (B) For supershift, binding reactions were carried out in the presence of antibodies against Sp1 or non-immune goat IgG (negative control). (C) ChIP analysis in HepG2 cells showed that GBE induced Sp1 to the indicated binding sites. Left panel, input control (for each group, 0.15 μg DNA was used for PCR); middle panel, Sp1 immunoprecipitation; and right panel, IgG immunoprecipitation (negative control). PCR was performed as described in Chromatin Immunoprecipitation Assay (ChIP) Section and products were run on a 2.0% agarose gel. D, G200, Q20, K20, I8 represent HepG2 cells incubated with 0.1% DMSO, 200 μg/ml GBE, 20 μg/ml quercetin, 20 μg/ml kaempferol, and 8 μg/ml isorhamnetin, respectively.