Figure 3
Wnt4 5′-flanking region contains a binding site for Pax8. A) Graphic output of the sequence analysis showing the conservation in different species of the core consensus sequences (in bold) of Pax8 binding sites. The sequence alignment was obtained using whole genome comparative analysis of the VISTA browser; B) Electromobility shift assays for Pax8 binding to putative recognition motifs in the Wnt4 promoter. Left panel, 32P-labeled oligo probes A and B were challenged with total protein extracts prepared from FRTL-5 cells (lane 2). Protein extracts of HeLa (lane 3) and Pax8-transfected HeLa cells (lane 4) were used as negative and positive control, respectively. Middle panel, the specificity of the complex observed with the FRTL-5 extract and probe A was tested by competition analysis, using increasing amount (from 25 to 100-fold molar excess) of unlabeled wild-type oligo A or mutated in the core sequence. Right panel, the FRTL-5 protein extract was incubated with the antibody against Pax8 or tubulin (as negative control), in a supershift EMSA with labeled probe A; C) Pax8 binding site A is necessary for the transcriptional activity of Wnt4 promoter. The deletion constructs 300Wnt4LUC and 260Wnt4LUC were transfected into FRTL-5 thyroid cells and the luciferase activity was determined. Data are from three independent experiments, each performed in duplicate and are expressed as the mean ± SD (P < 0,05).