Fig. 3.

Effect of 5-HT on the DNA-binding activity of nuclear proteins to the NHE3 core promoter region. Nucleotide sequence of the NHE3 core promoter region (bp −95 to −5) is shown and probes used for GMSAs are indicated (A). DNA-binding of nuclear proteins from untreated and serotonin treated cells to the probes A and B (B). Detailed analysis of DNA-binding characteristics of probe A and nuclear proteins from untreated and 5-HT treated cells (C). Lanes 1–5 and 6–9 show complex formation between probe A and nuclear proteins from untreated or serotonin-treated cells, respectively. The binding specificity of these complexes was examined by competition assays where excess unlabeled Sp1-specific probe was used (lane 5). The identities of the proteins present in these complexes were established by supershift assays and are indicated by slow migrating supershift bands (SS). Effects of serotonin on the nuclear expression of Sp1 and Sp3 in C2BBe1 cells (D). Nuclear proteins (20 μg/lane) were separated by 10% SDS-polyacrylamide gel electrophoresis and Western blots were performed. Sp1 and Sp3 were detected using Sp1 and Sp3 antibodies, respectively. The blots were re-probed with actin antibody. C, control; Ab, antibody; NP, nuclear proteins.