Fig. 3.

GATA-6 interacts with conserved binding site in AQP5 promoter. A: proximal −1,716-bp fragment of the 5′-flanking region of the AT1 cell-specific rat AQP5 gene contains 9 putative GATA-6-binding sites upstream of the transcription start site. The transcriptional start site is designated as −128 relative to the ATG (+1) (3). B: electrophoretic mobility shift assay (EMSA) was performed using nuclear extract (NE) from NIH 3T3 cells transfected with pcDNA3/GATA-6 and a ³³P-labeled oligonucleotide probe. A DNA-protein complex (arrow) is formed with probe 2 (spanning −1,197/−1,174 in the AQP5 promoter). Complexes are competed by 100× wild-type (Wt) but not mutated cold competitor (M1 and M2). C: soluble chromatin from MLE-15 cells was immunoprecipitated with GATA-6 antibody or IgG. Coprecipitated DNA was analyzed by PCR using a primer pair specific for the no. 2 conserved GATA-binding site in the AQP5 promoter/enhancer. Albumin was used as nonspecific binding control.