Fig. 5.

c-Fos or c-Jun binds to potential AP1 cis elements of Pgp promoter. A: electrophoretic mobility shift assay was performed using a double-stranded oligonucleotide as DIG-labeled proximal AP1a probe (−119/−98 bp) and nuclear extracts from untreated (control) Caco-2 cells or Caco-2 cells treated with LA CS (1:10 dilution, 24 h). Lane 1 depicts only probe. DNA-protein binding in control (lane 2) was significantly increased (lane 3) in response to LA CS. Competition experiments were performed in the presence of unlabeled cold AP1a oligonucleotide (lanes 4 and 5) and consensus AP1 oligonucleotide (lanes 6 and 7). DNA-protein complex was completely blocked in the presence of c-Fos antibody (2 μg; lanes 8 and 9) or partially blocked in the presence of c-Jun antibody (2 μg; lanes 10 and 11). B: DNA-protein complex was eliminated in the presence of an excess of cold unlabeled AP1a (lane 2) or consensus AP1 (lane 3) oligonucleotide or cold unlabeled AP1b oligonucleotide (lane 4), but not in the presence of consensus mutant AP1 oligonucleotide (lane 5) or unlabeled cold mutant AP1a oligonucleotide (M3; lane 8). Unlabeled cold mutant AP1a oligonucleotides M1 and M2 (lanes 6 and 7) eliminated the DNA-protein complex. + and −, Presence and absence of reaction components in the reaction mixture. Gels are representative of 3 separate experiments with similar results.