FIG. 6.

Demonstration by gel shift analysis of CREB and AP-1 activation in human intestinal epithelial cells (HT-29). Act activated CREB (A) and AP-1 (B) transcription factors in polarized human intestinal epithelial cells, as determined by gel shift assay. Nuclear extracts from polarized HT-29 cells treated on the apical side with Act (50 ng/ml; 0 and 30 min and 1, 2, and 4 h) were mixed with radiolabeled consensus oligonucleotides for CREB or AP-1 and subjected to nondenaturing 4% polyacrylamide gel electrophoresis (lanes 3 to 7). Nuclear extract from HT-29 cells treated for 4 h with Act was mixed with ∼50-fold excess unlabeled CREB or AP-1 consensus oligonucleotide (specific competition, lane 8) or NF-κB (nonspecific competition, lane 9) before the addition of the labeled oligonucleotide. HeLa cells extract was used as a positive control (PC, lane 1), and no extract was added as a negative control (NE, lane 2). The gel was dried and subjected to autoradiography.